14-3-3 Antagonists for the Prevention and Treatment of Arthritis

ABSTRACT

Methods for treating arthritis comprising 14-3-3 antagonists that are capable of specifically binding to extracellularly localized 14-3-3 eta and/or 14-3-3 gamma protein isoforms are provided. In preferred embodiments, the 14-3-3 antagonist is an inhibitory peptide or an anti-14-3-3 antibody. The 14-3-3 antagonists are also formulated in a pharmaceutical compositoin and used in a method for reducing matrix metalloprotease (MMP) expression in the synovial fluid of a patient, wherein the MMP is MMP-1 or MMP-3.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/990,520, filed 27 Nov. 2007, and U.S.Provisional Patent Application Ser. No. 61/077,123, filed 30 Jun. 2008,which are expressly incorporated herein in their entirety by reference.

FIELD OF THE INVENTION

The invention relates to the involvement of 14-3-3 proteins inarthritis, and compositions and methods for the prevention and treatmentof arthritis.

BACKGROUND OF THE INVENTION

Arthritis, or arthralgia, generally refers to inflammatory disorders ofthe joints of the body, and is usually accompanied by pain, swelling andstiffness. Arthritis may result from any of several causes includinginfection, trauma, degenerative disorders, metabolic disorders ordisturbances or other unknown etiologies. Osteoarthritis (OA) is acommon form of arthritis that may occur following trauma to a joint,following an infection of a joint or simply as a result of aging.Osteoarthritis is also known as degenerative joint disease. Rheumatoidarthritis (RA) is traditionally considered a chronic, inflammatoryautoimmune disorder that causes the immune system to attack the joints.It is a disabling and painful inflammatory condition which can lead tosubstantial loss of mobility due to pain and joint destruction.Ankylosing spondylitis (AS) is a chronic, painful, degenerativeinflammatory arthritis primarily affecting the spine and sacroiliacjoints, causing eventual fusion of the spine.

The body's articulating joints are called synovial joints, and eachsynovial joint generally comprises the opposing ends of two adjacentbones. The ends of the bones are encased in cartilage tissue while theentire joint area is encased in a protective soft tissue called synoviumwhich comprises synovial membrane. The synovial membrane produces andreleases a lubricating synovial fluid into cavities within the joint. Innormal joints, the volume of synovial fluid is quite small. In additionto its lubricating function, synovial fluid also acts as a reservoir forsolutes and a few resting mononuclear and synovial cells.

The synovium can become irritated and thickened in response to manyinsults believed to promote arthritis, including trauma to the jointand/or malfunction of the body's immune system. The consequences of suchinsults include excessive production and release of synovial fluid intothe joint, thereby causing swelling within and about the joint area. Theincreased volumes are typically accompanied by increased concentrationsin the synovial fluid of fibroblast-like synoviocyte cells (FLS cells),pro-inflammatory cytokines such as interleukin-1 (IL-1) and tumornecrosis factor (TNF-alpha), histamine proteins and peptides, anddegradative enzymes such as matrix metalloproteases (MMPs). The FLScells comprise about two-thirds of the synovial cells in normal synovialfluid, have well-defined secretory systems, and under conditions oftrauma or inflammation commonly secrete large amounts of MMPs into thesynovial fluid, specifically MMP-1, 3, 8, 9, 10, 11 and 13. MMP-1 andMMP-3 are considered to have significant roles in the progressivestructural damage of cartilage and underlying bone tissues comprisingjoints. Known factors that activate FLS cells to produce MMP-1 and MMP-3include IL-1 and TNF-alpha.

The causative agents for RA, SA and OA are currently not well-defined.However, the physiological events associated with progression of thedisease, from prolonged periods of swelling and inflammation caused byexcessive synovial fluid accumulation in the joints, through degradationand deterioration of the cartilage and underlying bone tissues bydegradative enzyme activities, and the accompanying FLS cellproliferation into bone which results in permanent structural damage,are known. If detected early enough, the potential long-term deleteriouseffects of disease can be reversed, or at least minimized, withappropriate physical and medical therapies. Accordingly, considerableefforts have been placed on the identification of suitable biomarkersfor early identification of arthritis. To this end, Kilani et al. (2007,J. Rheum. 34: 1650-1657; WO 2007/128132) have reported that two membersof the 14-3-3 protein family, particularly 14-3-3 eta and 14-3-3 gamma,are present within the synovial fluid and serum of patients witharthritis, and these isoforms are directly correlated with the levels ofMMP-1 and MMP-3 in the synovial fluid and serum.

14-3-3 proteins are a family of conserved intracellular regulatorymolecules that are ubiquitously expressed in eukaryotes. 14-3-3 proteinshave the ability to bind a multitude of functionally diverse signalingproteins, including kinases, phosphatases, and transmembrane receptors.Indeed, more than 100 signaling proteins have been reported as 14-3-3ligands. 14-3-3 proteins may be considered evolved members of theTetratrico Peptide Repeat superfamily. They generally have 9 or 10 alphahelices, and usually form homo- and/or hetero-dimer interactions alongtheir amino-termini helices. These proteins contain a number of knowndomains, including regions for divalent cation interaction,phosphorylation & acetylation, and proteolytic cleavage, among others.There are seven distinct genetically encoded isoforms of the 14-3-3proteins that are known to be expressed in mammals, with each isoformcomprising between 242-255 amino acids. The seven 14-3-3 proteinisoforms are designated as 14-3-3 α/β (alpha/beta), 14-3-3 δ/ζ(delta/zeta), 14-3-3ε (epsilon), 14-3-3γ (gamma), 14-3-3η (eta), 14-3-3τ/θ (tau/theta), and 14-3-3σ (sigma/stratifin).

SUMMARY OF THE INVENTION

The invention stems in part from the findings that (i) 14-3-3 protein isaberrantly localized in the extracellular synovial space in arthritis,(ii) such extracellular 14-3-3 protein can induce effectors ofarthritis, and (iii) 14-3-3 antagonists directed to such extracellular14-3-3 proteins can reduce the effectors of arthritis.

In one aspect, the invention provides methods of treating arthritis. Ina preferred embodiment, the invention provides methods of treating adisease selected from the group consisting of ankylosing spondylitis,Behçet's Disease, diffuse idiopathic skeletal hyperostosis (DISH),Ehlers-Danlos Syndrome (EDS), Felty's Syndrome, fibromyalgia, gout,infectious arthritis, juvenile arthritis, lupus, mixed connective tissuedisease (MCTD), osteoarthritis, Paget's Disease, polymyalgia rheumatica,polymyositis and dermatomyositis, pseudogout, psoriatic arthritis,Raynaud's Phenomenon, reactive arthritis, rheumatoid arthritis,scleroderma, Sjögren's Syndrome, Still's Disease, and Wegener'sgranulomatosis.

The methods involve administration of a 14-3-3 antagonist to an affectedpatient, wherein the 14-3-3 antagonist is targeted to 14-3-3 proteinthat is localized extracellularly. In a preferred embodiment, the 14-3-3protein is 14-3-3 eta or 14-3-3 gamma.

The 14-3-3 antagonists used may be prior art compositions, or novelcompositions disclosed herein. Therapeutic compositions are formulatedand administration is such that the 14-3-3 antagonist so delivered isavailable to engage extracellular 14-3-3 protein. In one embodiment, the14-3-3 antagonist is a peptide or an anti-14-3-3 antibody.

In a preferred embodiment, the 14-3-3 antagonist used is capable ofinhibiting the induction of MMP by a 14-3-3 protein to which it binds.Preferably, the MMP is selected from the group consisting of MMP-1, 3,8, 9, 10, 11 and 13, with MMP-1 and MMP-3 being especially preferred.

In one embodiment, the method involves a combination treatment, whereinat least one other therapeutic agent is administered in addition to oneor more 14-3-3 antagonists. In a preferred embodiment, the therapeuticagent is selected from the group consisting of disease-modifyingantirheumatic drugs (DMARDs) and disease modifying osteoarthritis drugs(DMOADs; for example, see Loeser, Reumatologia, 21:104-106, 2005). In anespecially preferred embodiment, one or more anti-14-3-3 antagonists isadministered in combination with at least one agent selected from thegroup consisting of anti-TNFα antibody, anti-IL-1 antibody, anti-CD4antibody, anti-CTLA4 antibody, anti-IL-6 antibody, anti-CD20 antibody,leflunomide, sulfasalazine, and methotrexate.

In one embodiment, a 14-3-3 antagonist is administered in the form of anencoding nucleic acid that is expressed to deliver the 14-3-3antagonist.

In one embodiment, the method involves administering to a patient a cellthat delivers a 14-3-3 antagonist. In a preferred embodiment, the 14-3-3antagonist so delivered is a peptide or an anti-14-3-3 antibody. In apreferred embodiment, the cell is a fibroblast or an FLS cell.

In one aspect, the invention provides prophylactic methods forpreventing the development of arthritis in a subject at risk ofdeveloping arthritis. The methods comprise administering to the subjectone or more 14-3-3 antagonists.

In one aspect, the invention provides methods for reducing the damage toa joint injured by trauma. The methods comprise administering one ormore 14-3-3 antagonists to a subject having a joint injured by trauma.In one embodiment, a 14-3-3 antagonist is administered as a component ofa combination therapy described herein.

In one aspect, the invention provides methods of decreasing MMPexpression. In one embodiment, the MMP expression to be decreased is inthe synovium. The methods comprise delivering one or more 14-3-3antagonists to a tissue or compartment in which MMP producing cells arepresent, wherein the MMP producing cells are responsive to 14-3-3proteins to which the 14-3-3 antagonists bind. Delivery may be direct tothe affected tissue or compartment, or indirect. In a preferredembodiment, the responsive cells are fibroblasts or FLS cells.

In a preferred embodiment, the MMP expression that is to be decreased isMMP expression that is associated with arthritis.

In a preferred embodiment, the MMP expression that is to be decreased isthat of an MMP selected from the group consisting of MMP-1, 3, 8, 9, 10,11 and 13. In an especially preferred embodiment, the MMP expressionthat is to be decreased is that of MMP-1 or MMP-3.

In one aspect, the invention provides methods of inhibiting MMPinduction by a 14-3-3 protein. Inhibition may be partial or complete.The methods comprise delivering one or more 14-3-3 antagonists to atissue or compartment in which MMP producing cells are present, whereinthe MMP producing cells are responsive to 14-3-3 proteins to which the14-3-3 antagonists specifically bind. Delivery may be direct to theaffected tissue or compartment, or indirect. In a preferred embodiment,the one or more 14-3-3 antagonists are administered to the synovium. Ina preferred embodiment, the responsive cells are fibroblasts or FLScells.

In a preferred embodiment, the MMP induction that is to be inhibited isthat of an MMP which is upregulated in arthritis.

In a preferred embodiment, the MMP induction that is to be inhibited isthat of an MMP selected from the group consisting of MMP-1, 3, 8, 9, 10,11 and 13. In an especially preferred embodiment, the MMP induction thatis to be inhibited is that of MMP-1 or MMP-3.

In one aspect, the invention provides methods of decreasing jointswelling in a subject. The methods comprise administering one or more14-3-3 antagonists to an affected subject.

In one aspect, the invention provides methods of decreasing cartilagedegradation in a subject. The methods comprise administering one or more14-3-3 antagonists to an affected subject.

In one aspect, the invention provides methods of decreasing bonedegradation in a subject. The methods comprise administering one or more14-3-3 antagonists to an affected subject.

In one aspect, the invention provides methods of decreasingpro-inflammatory cytokine accumulation in synovial fluid. The methodscomprise administering one or more 14-3-3 antagonists to an affectedsubject.

For methods involving administration of a 14-3-3 antagonist to anaffected subject, in a preferred embodiment, intracapsular delivery ofantagonist is used. In another embodiment, systemic delivery ofantagonist is used. The therapeutic compositions are formulated andadministration is such that the 14-3-3 antagonist so delivered isavailable to engage extracellularly localized 14-3-3 protein.

In one embodiment, the 14-3-3 antagonist is a peptide. In a preferredembodiment, the peptide comprises the amino acid sequence designated“R-18”. In another preferred embodiment, the peptide consistsessentially of the R-18 sequence. In one embodiment, the peptidecomprises multiple iterations of the R-18 sequence. In another preferredembodiment, the peptide binds to a region of 14-3-3 protein that iscapable of binding to R-18. In another preferred embodiment, the peptidebinds to a region of a 14-3-3 protein that is capable of binding to anintracellular 14-3-3 binding partner, preferably Raf. In one embodiment,the peptide binds to a 14-3-3 protein without disrupting binding of the14-3-3 protein to an intracellular 14-3-3 binding partner.

In one embodiment, the 14-3-3 antagonist is a phosphopeptide.

In one embodiment, the 14-3-3 antagonist is a mode I phosphopeptide, asis known in the art.

In another embodiment, the 14-3-3 antagonist is a mode IIphosphopeptide, as is known in the art.

In one embodiment, the 14-3-3 antagonist is an anti-14-3-3 antibody. Inone embodiment, the anti-14-3-3 antibody is a pan 14-3-3 antibody. Inanother embodiment, the anti-14-3-3 antibody is capable ofdistinguishing between 14-3-3 isoforms. In a preferred embodiment, theanti-14-3-3 antibody specifically binds to a peptide selected from thegroup consisting of 14-3-3 loop peptides, 14-3-3 helix peptides, andnon-helix 14-3-3 peptides.

In one embodiment, the anti-14-3-3 antibody is an anti-14-3-3 gammaantibody. In a preferred embodiment, the anti-14-3-3 gamma antibodybinds to a 14-3-3 peptide comprising a segment of the amino acidsequence set forth in SEQ ID NO:64. In a preferred embodiment, thesegment is at least 6, more preferably at least 7, more preferably atleast 8 amino acids in length.

In a preferred embodiment, an anti-14-3-3 gamma antibody binds to a14-3-3 peptide that is a 14-3-3 gamma loop peptide. In a preferredembodiment, the 14-3-3 gamma loop peptide comprises an amino acidsequence selected from the group consisting of SEQ ID NOs:44-49. Inanother embodiment, the 14-3-3 gamma loop peptide consists essentiallyof an amino acid sequence selected from the group consisting of SEQ IDNOs:44-49. In another embodiment, an anti-14-3-3 gamma antibody binds toa region of 14-3-3 gamma that overlaps with an amino acid sequencecorresponding to a sequence selected from the group consisting of SEQ IDNOs:44-49.

In another preferred embodiment, an anti-14-3-3 gamma antibody binds toa 14-3-3 peptide that is a 14-3-3 gamma helix peptide. In a preferredembodiment, the 14-3-3 gamma helix peptide comprises an amino acidsequence selected from the group consisting of SEQ ID NOs:33-43. Inanother embodiment, the 14-3-3 gamma helix peptide consists essentiallyof an amino acid sequence selected from the group consisting of SEQ IDNOs:33-43. In another embodiment, an anti-14-3-3 gamma antibody binds toa region of 14-3-3 gamma that overlaps with an amino acid sequencecorresponding to a sequence selected from the group consisting of SEQ IDNOs:33-43.

In another preferred embodiment, an anti-14-3-3 gamma antibody binds toa 14-3-3 peptide that is a non-helix 14-3-3 gamma peptide. In apreferred embodiment, the non-helix 14-3-3 gamma peptide comprises anamino acid sequence selected from the group consisting of SEQ IDNOs:50-62. In another embodiment, the non-helix 14-3-3 gamma peptideconsists essentially of an amino acid sequence selected from the groupconsisting of SEQ ID NOs:50-62. In another embodiment, an anti-14-3-3gamma antibody binds to a region of 14-3-3 gamma that overlaps with anamino acid sequence corresponding to a sequence selected from the groupconsisting of SEQ ID NOs:50-62.

In one embodiment, the anti-14-3-3 antibody is an anti-14-3-3 etaantibody. In a preferred embodiment, the anti-14-3-3 eta antibody bindsto a 14-3-3 peptide comprising a segment of the amino acid sequence setforth in SEQ ID NO:63. In a preferred embodiment, the segment is atleast 6, more preferably at least 7, more preferably at least 8 aminoacids in length.

In one embodiment, an anti-14-3-3 eta antibody of the invention does notbind to an epitope located at the N-terminus of the human 14-3-3 etaprotein.

In a preferred embodiment, an anti-14-3-3 eta antibody binds to a 14-3-3peptide that is a 14-3-3 eta loop peptide. In a preferred embodiment,the 14-3-3 eta loop peptide comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs:11-16. In another embodiment,the 14-3-3 eta loop peptide consists essentially of an amino acidsequence selected from the group consisting of SEQ ID NOs:11-16. Inanother embodiment, an anti-14-3-3 eta antibody binds to a region of14-3-3 eta that overlaps with an amino acid sequence corresponding to asequence selected from the group consisting of SEQ ID NOs:11-16.

In another preferred embodiment, an anti-14-3-3 eta antibody binds to a14-3-3 peptide that is a 14-3-3 eta helix peptide. In a preferredembodiment, the 14-3-3 eta helix peptide comprises an amino acidsequence selected from the group consisting of SEQ ID NOs:1-10. Inanother embodiment, the 14-3-3 eta helix peptide consists essentially ofan amino acid sequence selected from the group consisting of SEQ IDNOs:1-10. In another embodiment, an anti-14-3-3 eta antibody binds to aregion of 14-3-3 eta that overlaps with an amino acid sequencecorresponding to a sequence selected from the group consisting of SEQ IDNOs:1-10.

In another preferred embodiment, an anti-14-3-3 eta antibody binds to a14-3-3 peptide that is a non-helix 14-3-3 eta peptide. In a preferredembodiment, the non-helix 14-3-3 eta peptide comprises an amino acidsequence selected from the group consisting of SEQ ID NOs:17-32. Inanother embodiment, the non-helix 14-3-3 eta peptide consistsessentially of an amino acid sequence selected from the group consistingof SEQ ID NOs:17-32. In another embodiment, an anti-14-3-3 eta antibodybinds to a region of 14-3-3 eta that overlaps with an amino acidsequence corresponding to a sequence selected from the group consistingof SEQ ID NOs:17-32.

In an especially preferred embodiment, an anti-14-3-3 eta antibody ofthe invention binds to an amino acid sequence selected from the groupconsisting of LDKFLIKNSNDF (SEQ ID NO:30), KKLEKVKAYR (SEQ ID NO:31),and KNSVVEASEAAYKEA (SEQ ID NO:32).

Exemplary 14-3-3 eta loop, helix, and non-helix peptides are disclosedin Table 1 herein. Notably, SEQ ID NO:30 varies from corresponding14-3-3 eta sequence in that a cysteine occurring in 14-3-3 eta sequencehas been replaced by serine to avoid disulfide bond formation. In oneembodiment, the invention provides antibodies that also bind to thenatural 14-3-3 sequence correlate of SEQ ID NO:30 comprising a cysteine.In one embodiment, the invention provides antibodies capable of bindingto peptide sequences that vary from those listed in the epitope tablesherein by substitution of serine for cysteine.

In a preferred embodiment, an anti-14-3-3 antibody is capable ofdiscriminating between 14-3-3 protein isoforms.

In a preferred embodiment, an anti-14-3-3 antibody is a monoclonalantibody.

In a preferred embodiment, an anti-14-3-3 antibody is a humanizedantibody.

In one aspect, the invention provides novel 14-3-3 antagonists. In oneembodiment, the invention provides novel 14-3-3 antagonist peptides. Inanother embodiment, the invention provides novel anti-14-3-3 antibodies.In one aspect, the invention provides methods of making 14-3-3antagonists.

In one aspect, the invention provides nucleic acids encoding 14-3-3antagonists that are peptides or antibodies. Also provided are vectors,including expression vectors, comprising such nucleic acids. Alsoprovided are host cells comprising such nucleic acids and host cellscomprising such vectors. Also provided are methods of making a 14-3-3antagonist, which comprise the use of such host cells.

In one aspect, the invention provides cells capable of producing 14-3-3antagonists.

In one embodiment, the cell is a hybridoma, and the 14-3-3 antagonist isan anti-14-3-3 antibody.

In another embodiment, the cell is a genetically modified fibroblast orFLS cell, and the 14-3-3 antagonist is a peptide or an anti-14-3-3antibody.

In one aspect, the invention provides methods of screening for a 14-3-3antagonist. In one embodiment, the methods comprise screening candidateagents for the ability to inhibit binding of a 14-3-3 eta protein or14-3-3 gamma protein to 14-3-3 eta ligand or a 14-3-3 gamma ligand,respectively. In one embodiment, the ligand is a 14-3-3 antagonistpeptide. In one embodiment, the ligand is an anti-14-3-3 antibody. Inone embodiment, the ligand is an intracellular 14-3-3 binding partner.In one embodiment, the methods comprise analyzing the ability of thecandidate agent to inhibit induction of MMP by a 14-3-3 protein.

In one embodiment, a 14-3-3 antagonist competitively inhibits thebinding of a 14-3-3 eta protein or a 14-3-3 gamma protein to ananti-14-3-3 antibody or a 14-3-3 antagonist peptide disclosed herein. Inone embodiment, the 14-3-3 antagonist is a small molecule chemicalcomposition.

In a preferred embodiment, a 14-3-3 antagonist binds to a region of14-3-3 protein that is capable of binding to R-18. In a preferredembodiment, a 14-3-3 antagonist binds to a region of 14-3-3 protein thatis capable binding to an intracellular 14-3-3 binding partner,preferably Raf. In one embodiment, a 14-3-3 antagonist binds to a 14-3-3protein without inhibiting the binding of an intracellular 14-3-3binding partner.

In one aspect, the invention provides pharmaceutical compositions forthe treatment of arthritis. The pharmaceutical compositions comprise oneor more 14-3-3 antagonists. The pharmaceutical compositions areformulated to provide for engagement of extracellular 14-3-3 protein bythe 14-3-3 antagonist.

In one aspect, the invention provides methods for preparing a medicamentuseful for treating arthritis. Such a medicament comprises one or more14-3-3 antagonists. The medicament is formulated to provide forengagement of extracellular 14-3-3 protein by the 14-3-3 antagonist.

In one aspect, the invention accordingly involves the use of a 14-3-3antagonist, such as a 14-3-3 antagonist that is capable of specificallybinding to an extracellularly-localized 14-3-3 protein and inhibitingthe activity of the 14-3-3 protein, for treating arthritis or toformulate a medicament for treating arthritis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. ELISA: Test Bleed Titration of Mouse Anti-AUG1-CLDK Immune Serum(after 2nd boost) on AUG1-CLDK-BSA Antigen (IgG response only).

FIG. 2. ELISA: Test Bleed Titration of Mouse Anti-AUG2-KKLE Immune Serum(after 2nd boost) on AUG2-KKLE-BSA antigen (IgG response only).

FIG. 3. ELISA: Test Bleed Titration of Mouse Anti-AUG3-CKNS Immune Serum(after 2nd boost) on AUG3-CKNS-BSA Antigen (IgG response only).

FIG. 4. Sequence alignment for various 14-3-3 protein isoforms.

FIGS. 5A & B. R-18 interacts with extracellular 14-3-3 protein andinhibits induction of MMP-1 expression induced by extracellular 14-3-3protein.

FIG. 6. Western Blot showing cell lysate-derived 14-3-3 eta protein andhuman recombinant 14-3-3 eta immunoprecipated by monoclonal antibodyraised against full length human recombinant 14-3-3 eta.

FIG. 7. Western Blot showing cell lysate-derived 14-3-3 eta protein andhuman recombinant 14-3-3 eta immunoprecipated by monoclonal antibodyraised against a human 14-3-3 eta peptide fragment 142-158 SEQ ID NO:24from a non-helical region of the protein.

FIG. 8. ELISA: Test Bleed Titration of Mouse anti-14-3-3 eta Immune Sera(after 2nd boost) on 14-3-3 eta Antigen (IgG response only)

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods of treating arthritis, including methodsof treating ankylosing spondylitis, Behçet's Disease, diffuse idiopathicskeletal hyperostosis (DISH), Ehlers-Danlos Syndrome (EDS), Felty'sSyndrome, fibromyalgia, gout, infectious arthritis, juvenile arthritis,lupus, mixed connective tissue disease (MCTD), osteoarthritis, Paget'sDisease, polymyalgia rheumatica, polymyositis and dermatomyositis,pseudogout, psoriatic arthritis, Raynaud's Phenomenon, reactivearthritis, rheumatoid arthritis, scleroderma, Sjögren's Syndrome,Still's Disease, and Wegener's granulomatosis.

As used herein, ‘arthritis’ or ‘arthralgia’ refer to an inflammatorydisorder of the joints of the body. Pain, swelling, stiffness anddifficulty of movement are frequently associated with arthritisdiseases. Arthritis may result from any of several causes includinginfection, trauma, degenerative disorders, metabolic disorders ordisturbances or other unknown etiologies.

The progression or severity of arthritis in a patient may be measured orquantified using techniques known in the art. As an example, a “DiseaseActivity Score” (DAS) may be utilized to measure the activity or stateof arthritis in a patient. DAS is one of several standards or scoresused in clinical practice. A calculation of a DAS may include thefollowing parameters: Number of joints tender to the touch (TEN), numberof swollen joints (SW), erythrocyte sedimentation rate (ESR) and patientassessment of disease activity (VAS). Alternatively, a DAS may includeC-reactive protein marker assessment (CRP) (Skogh T et al 2003. AnnRheum Dis 62:681-682). Alternately, one may utilize diagnosticbiomarkers, including 14-3-3 eta and/or gamma, to measure the presenceor absence of disease, or to determine disease severity.

In the present description the terms “treatment” or “treat” refer toboth prophylactic or preventative treatment as well as curative ordisease modifying treatment, including treatment of a patient at risk ofcontracting the disease or suspected to have contracted the disease aswell as patients who are ill or have been diagnosed as suffering from adisease or medical condition, and includes suppression of clinicalrelapse.

The methods involve administration of one or more 14-3-3 antagonists. A14-3-3 antagonist of the invention binds to a 14-3-3 protein,particularly 14-3-3 eta or gamma, and antagonizes the activity thereof.

As used herein, an ‘isoform’ refers to two or more functionally similarproteins that have a similar but not identical amino acid sequence andare either encoded by different genes or by different RNA transcripts,primary or processed, from the same gene.

Reference to a 14-3-3 eta protein or a 14-3-3 gamma protein may includefragments thereof. For example, in one embodiment the invention providesmethods of screening for a 14-3-3 antagonist which, in a preferredembodiment, comprise screening candidate agents for the ability toinhibit binding of a 14-3-3 ligand to a 14-3-3 protein. It will beunderstood that an appropriate fragment of a 14-3-3 protein can be usedin the assay.

An example of a 14-3-3 antagonist is the R18 inhibitory peptide (Wang etal. 1999—REF 35). The R18 peptide, also referred to herein as ‘R18’, isa small peptide which is capable of blocking the association of 14-3-3proteins with Raf-1. Other examples of peptides that bind to 14-3-3proteins are known (see, for example, Wang et al. 1999—REF 35, infra;Yaffe et al., Cell, 91:961-971, 1997; Shaw et al., U.S. Pat. No.5,948,765; Petosa et al., JBC 273:16305-16310, 1998; Fu et al., US2004/0152630). These and others when formulated to engage aberrantlylocalized extracellular 14-3-3 protein may find utility in the presentinvention as therapeutics for the treatment of arthritis.

“Antibody” refers to a composition comprising a protein that bindsspecifically to a corresponding antigen and has a common, generalstructure of immunoglobulins. The term antibody specifically coverspolyclonal antibodies, monoclonal antibodies, dimers, multimers,multispecific antibodies (e.g., bispecific antibodies), and antibodyfragments, so long as they exhibit the desired biological activity.Antibodies may be murine, human, humanized, chimeric, or derived fromother species. Typically, an antibody will comprise at least two heavychains and two light chains interconnected by disulfide bonds, whichwhen combined form a binding domain that interacts with an antigen. Eachheavy chain is comprised of a heavy chain variable region (VH) and aheavy chain constant region (CH). The heavy chain constant region iscomprised of three domains, CH1, CH2 and CH3, and may be of the mu,delta, gamma, alpha or epsilon isotype. Similarly, the light chain iscomprised of a light chain variable region (VL) and a light chainconstant region (CL). The light chain constant region is comprised ofone domain, CL, which may be of the kappa or lambda isotype. The VH andVL regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). Each VHand VL is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (Clq)of the classical complement system. The heavy chain constant regionmediates binding of the immunoglobulin to host tissue or host factors,particularly through cellular receptors such as the Fc receptors (e.g.,FcγRI, FcγRII, FcγRIII, etc.). As used herein, antibody also includes anantigen binding portion of an immunoglobulin that retains the ability tobind antigen. These include, as examples, F(ab), a monovalent fragmentof VL CL and VH CH antibody domains; and F(ab′)₂ fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region. The term antibody also refers to recombinant singlechain Fv fragments (scFv) and bispecific molecules such as, e.g.,diabodies, triabodies, and tetrabodies (see, e.g., U.S. Pat. No.5,844,094).

Antibodies may be produced and used in many forms, including antibodycomplexes. As used herein, the term “antibody complex” refers to acomplex of one or more antibodies with another antibody or with anantibody fragment or fragments, or a complex of two or more antibodyfragments. Antibody complexes include multimeric forms of anti-14-3-3antibodies such as homoconjugates and heteroconjugates as well as othercross-linked antibodies as described herein.

“Antigen” is to be construed broadly and refers to any molecule,composition, or particle that can bind specifically to an antibody. Anantigen has one or more epitopes that interact with the antibody,although it does not necessarily induce production of that antibody.

The terms “cross-linked”, “cross-linking” and grammatical equivalentsthereof, refer to the attachment of two or more antibodies to formantibody complexes, and may also be referred to as multimerization.Cross-linking or multimerization includes the attachment of two or moreof the same antibodies (e.g. homodimerization), as well as theattachment of two or more different antibodies (e.g.heterodimerization). Those of skill in the art will also recognize thatcross-linking or multimerization is also referred to as forming antibodyhomoconjugates and antibody heteroconjugates. Such conjugates mayinvolve the attachment of two or more monoclonal antibodies of the sameclonal origin (homoconjugates) or the attachment of two or moreantibodies of different clonal origin (also referred to asheteroconjugates or bispecific). Antibodies may be crosslinked bynon-covalent or covalent attachment. Numerous techniques suitable forcross-linking will be appreciated by those of skill in the art.Non-covalent attachment may be achieved through the use of a secondaryantibody that is specific to the primary antibody species. For example,a goat anti-mouse (GAM) secondary antibody may be used to cross-link amouse monoclonal antibody. Covalent attachment may be achieved throughthe use of chemical cross-linkers.

“Epitope” refers to a determinant capable of specific binding to anantibody. Epitopes are chemical features generally present on surfacesof molecules and accessible to interaction with an antibody. Typicalchemical features are amino acids and sugar moieties, havingthree-dimensional structural characteristics as well as chemicalproperties including charge, hydrophilicity, and lipophilicity.Conformational epitopes are distinguished from non-conformationalepitopes by loss of reactivity with an antibody following a change inthe spatial elements of the molecule without any change in theunderlying chemical structure.

“Humanized antibody” refers to an immunoglobulin molecule containing aminimal sequence derived from non-human immunoglobulin. Humanizedantibodies include human immunoglobulins (recipient antibody) in whichresidues from a complementary determining region (CDR) of the recipientare replaced by residues from a CDR of a non-human species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity and capacity. In some instances, Fv framework residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, a humanized antibody will comprise substantiallyall of at least one, and typically two, variable domains, in which allor substantially all of the CDR regions correspond to those of anon-human immunoglobulin and all or substantially all of the framework(FR) regions are those of a human immunoglobulin consensus sequence. Ahumanized antibody will also encompass immunoglobulins comprising atleast a portion of an immunoglobulin constant region (Fc), generallythat of a human immunoglobulin (Jones et al., Nature 321:522-525 (1986);Reichmann et al, Nature 332:323-329 (1988)).

“Immunogen” refers to a substance, compound, or composition whichstimulates the production of an immune response.

The term “immunoglobulin locus” refers to a genetic element or set oflinked genetic elements that comprise information that can be used by aB cell or B cell precursor to express an immunoglobulin polypeptide.This polypeptide can be a heavy chain polypeptide, a light chainpolypeptide, or the fusion of a heavy and a light chain polypeptide. Inthe case of an unrearranged locus, the genetic elements are assembled bya B cell precursor to form the gene encoding an immunoglobulinpolypeptide. In the case of a rearranged locus, a gene encoding animmunoglobulin polypeptide is contained within the locus.

“Isotype” refers to an antibody class defined by its heavy chainconstant region. Heavy chains are generally classified as gamma, mu,alpha, delta, epsilon and designated as IgG, IgM, IgA, IgD, and IgE.Variations within each isotype are categorized into subtypes, forexample subtypes of IgG are divided into IgG1, IgG2, IgG3, and IgG4,while IgA is divided into IgA1 and IgA2. The IgY isotype is specific tobirds.

“Monoclonal antibody” or “monoclonal antibody composition” refers to apreparation of antibody molecules of single molecular composition. Amonoclonal antibody composition displays a single binding specificityand affinity for a particular epitope.

The term “human monoclonal antibody” includes antibodies displaying asingle binding specificity which have variable and/or constant regions(if present) derived from human immunoglobulin sequences. In oneembodiment, the human monoclonal antibodies are produced by a hybridomawhich includes a B cell obtained from a transgenic non-human animal,e.g., a transgenic mouse, having a genome comprising a human heavy chaintransgene and a light chain transgene, fused to an immortalized cell.

“Single chain Fv” or “scFv” refers to an antibody comprising the VH andVL regions of an antibody, wherein these domains are present in a singlepolypeptide chain. Generally, an scFv further comprises a polypeptidelinker between the VH and VL domains which enables the scFv to form thedesired structure for antigen binding.

“Subject” or “patient” are used interchangeably and refer to, exceptwhere indicated, mammals such as humans and non-human primates, as wellas rabbits, rats, mice, goats, pigs, and other mammalian species.

“Recombinant antibody” refers to all antibodies produced by recombinanttechniques. These include antibodies obtained from an animal that istransgenic for the immunoglobulin locus, antibodies expressed from arecombinant expression vector, or antibodies created, prepared, andexpressed by splicing of any immunoglobulin gene sequence to any othernucleic acid sequence.

Anti-14-3-3 Antibodies

In one aspect, the invention provides novel anti-14-3-3 antibodies thatbind specifically to 14-3-3 eta or 14-3-3 gamma protein. Preferably, ananti-14-3-3 antibody of the invention is capable of specifically bindingto 14-3-3 protein in its natural 3-D configuration. By specificallybinding to a 14-3-3 protein in its “natural configuration” is meant anability to bind to 14-3-3 protein as encountered in vivo. This may beevidenced, for example, by the ability of antibody to immunoprecipitate14-3-3 eta protein from a biological sample.

In a preferred embodiment, an anti-14-3-3 eta antibody of the inventionis capable of binding to 14-3-3 protein that is aberrantly localized inthe extracellular synovial space in arthritis. This may be evidenced,for example, by immunoprecipitation of 14-3-3 protein present in asynovial fluid sample from a patient having arthritis.

In a preferred embodiment, an anti-14-3-3 antibody of the invention iscapable of discriminating between 14-3-3 protein isoforms. Suchantibodies have an ability to bind specifically to a particular 14-3-3protein isoform and bind preferentially to that isoform over other14-3-3 protein isoforms under the same conditions. This may beevidenced, for example, using an ELISA assay, which may be done using,for example, supernatant from hybridoma clones. A control (e.g.,pre-immune serum) is preferably used. A “selective” antibody is capableof recognizing a particular 14-3-3 isoform and generating a highersignal against that isoform as compared to other isoforms, preferably atleast a 1.5 fold, more preferably at least a 2 fold higher signal ascompared to other isoforms. In a preferred embodiment, a selectiveantibody has an ability to selectively immunoprecipitate the particular14-3-3 eta as compared to other 14-3-3 isoforms.

In a preferred embodiment, the anti-14-3-3 antibody exhibits suchselectivity for 14-3-3 eta protein over 14-3-3 alpha, beta, delta,epsilon, gamma, tau, and zeta proteins. This may be evidenced, forexample, by ELISA.

In another preferred embodiment, the anti-14-3-3 antibody exhibits suchselectivity for 14-3-3 gamma protein over 14-3-3 alpha, beta, delta,epsilon, eta, tau, and zeta proteins. This may be evidenced, forexample, by ELISA.

In a preferred embodiment, an anti-14-3-3 antibody of the invention is a14-3-3 antagonist, though other anti-14-3-3 antibodies are alsocontemplated within the scope of the invention.

In a preferred embodiment, an anti-14-3-3 antibody is capable ofinhibiting the induction of MMP by 14-3-3 protein, particularly 14-3-3gamma or 14-3-3 eta. Preferably, the MMP is selected from the groupconsisting of MMP-1, 3, 8, 9, 10, 11 and 13, with MMP-1 and MMP-3 beingespecially preferred. Such capability may be determined by an in vitroassay or in vivo assay. As will be appreciated by one of skill in theart, the assays will be designed such that in the absence of anti-14-3-3antibody, the presence of 14-3-3 protein will result in the induction ofMMP. An ability to reduce this induction of MMP by 14-3-3 protein canevidence such a function-inhibiting capability for an anti-14-3-3antibody.

In one aspect, the invention provides anti-14-3-3 eta antibodies. In apreferred embodiment, the anti-14-3-3 eta antibody binds to a 14-3-3peptide comprising a segment of the amino acid sequence set forth in SEQID NO:63. In a preferred embodiment, the segment is at least 6, morepreferably at least 7, more preferably at least 8 amino acids in length.

In one embodiment, an anti-14-3-3 eta antibody of the invention does notbind to an epitope located at the N-terminus of the human 14-3-3 etaprotein.

In a preferred embodiment, an anti-14-3-3 eta antibody binds to a 14-3-3peptide that is a 14-3-3 eta loop peptide. In a preferred embodiment,the 14-3-3 eta loop peptide comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs:11-16. In another embodiment,the 14-3-3 eta loop peptide consists essentially of an amino acidsequence selected from the group consisting of SEQ ID NOs:11-16. Inanother embodiment, an anti-14-3-3 eta antibody binds to a region of14-3-3 eta that overlaps with an amino acid sequence corresponding to asequence selected from the group consisting of SEQ ID NOs:11-16.

In another preferred embodiment, an anti-14-3-3 eta antibody binds to a14-3-3 peptide that is a 14-3-3 eta helix peptide. In a preferredembodiment, the 14-3-3 eta helix peptide comprises an amino acidsequence selected from the group consisting of SEQ ID NOs:1-10. Inanother embodiment, the 14-3-3 eta helix peptide consists essentially ofan amino acid sequence selected from the group consisting of SEQ IDNOs:1-10. In another embodiment, an anti-14-3-3 eta antibody binds to aregion of 14-3-3 eta that overlaps with an amino acid sequencecorresponding to a sequence selected from the group consisting of SEQ IDNOs:1-10.

In another preferred embodiment, an anti-14-3-3 eta antibody binds to a14-3-3 peptide that is a non-helix 14-3-3 eta peptide. In a preferredembodiment, the non-helix 14-3-3 eta peptide comprises an amino acidsequence selected from the group consisting of SEQ ID NOs:17-32. Inanother embodiment, the non-helix 14-3-3 eta peptide consistsessentially of an amino acid sequence selected from the group consistingof SEQ ID NOs:17-32. In another embodiment, an anti-14-3-3 eta antibodybinds to a region of 14-3-3 eta that overlaps with an amino acidsequence corresponding to a sequence selected from the group consistingof SEQ ID NOs:17-32.

In an especially preferred embodiment, an anti-14-3-3 eta antibody ofthe invention binds to an amino acid sequence selected from the groupconsisting of LDKFLIKNSNDF (SEQ ID NO:30), KKLEKVKAYR (SEQ ID NO:31),and KNSVVEASEAAYKEA (SEQ ID NO:32).

Exemplary 14-3-3 eta loop, helix, and non-helix peptides are disclosedin Table 1 herein. Notably, SEQ ID NO:30 varies from corresponding14-3-3 eta sequence in that a cysteine occurring in 14-3-3 eta sequencehas been replaced by serine to avoid disulfide bond formation. In oneembodiment, the invention provides antibodies that also bind to thenatural 14-3-3 sequence correlate of SEQ ID NO:30 comprising a cysteine.In one embodiment, the invention provides antibodies capable of bindingto peptide sequences that vary from those listed in the epitope tablesherein by substitution of serine for cysteine.

In one aspect, the invention provides anti-14-3-3 gamma antibodies. In apreferred embodiment, the anti-14-3-3 gamma antibody binds to a 14-3-3peptide comprising a segment of the amino acid sequence set forth in SEQID NO:64. In a preferred embodiment, the segment is at least 6, morepreferably at least 7, more preferably at least 8 amino acids in length.

In a preferred embodiment, an anti-14-3-3 gamma antibody binds to a14-3-3 peptide that is a 14-3-3 gamma loop peptide. In a preferredembodiment, the 14-3-3 gamma loop peptide comprises an amino acidsequence selected from the group consisting of SEQ ID NOs:44-49. Inanother embodiment, the 14-3-3 gamma loop peptide consists essentiallyof an amino acid sequence selected from the group consisting of SEQ IDNOs:44-49. In another embodiment, an anti-14-3-3 gamma antibody binds toa region of 14-3-3 gamma that overlaps with an amino acid sequencecorresponding to a sequence selected from the group consisting of SEQ IDNOs:44-49.

In another preferred embodiment, an anti-14-3-3 gamma antibody binds toa 14-3-3 peptide that is a 14-3-3 gamma helix peptide. In a preferredembodiment, the 14-3-3 gamma helix peptide comprises an amino acidsequence selected from the group consisting of SEQ ID NOs:33-43. Inanother embodiment, the 14-3-3 gamma helix peptide consists essentiallyof an amino acid sequence selected from the group consisting of SEQ IDNOs:33-43. In another embodiment, an anti-14-3-3 gamma antibody binds toa region of 14-3-3 gamma that overlaps with an amino acid sequencecorresponding to a sequence selected from the group consisting of SEQ IDNOs:33-43.

In another preferred embodiment, an anti-14-3-3 gamma antibody binds toa 14-3-3 peptide that is a non-helix 14-3-3 gamma peptide. In apreferred embodiment, the non-helix 14-3-3 gamma peptide comprises anamino acid sequence selected from the group consisting of SEQ IDNOs:50-62. In another embodiment, the non-helix 14-3-3 gamma peptideconsists essentially of an amino acid sequence selected from the groupconsisting of SEQ ID NOs:50-62. In another embodiment, an anti-14-3-3gamma antibody binds to a region of 14-3-3 gamma that overlaps with anamino acid sequence corresponding to a sequence selected from the groupconsisting of SEQ ID NOs:50-62.

Monoclonal Antibodies, Hybridomas, and Methods of Making the Same

The present disclosure provides monoclonal antibodies that specificallybind to 14-3-3 eta protein, as well as monoclonal antibodies thatspecifically bind to 14-3-3 gamma protein. Also provided are hybridomacell lines capable of producing such antibodies.

In one aspect, the invention provides monoclonal anti-14-3-3 etaantibodies that bind to a 14-3-3 eta loop, helix, or non-helix peptide.In a preferred embodiment, the peptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs:1-32. In an especiallypreferred embodiment, the invention provides anti-14-3-3 eta monoclonalantibodies that specifically bind to an amino acid sequence selectedfrom the group consisting of LDKFLIKNSNDF (SEQ ID NO:30), KKLEKVKAYR(SEQ ID NO:31), and KNSVVEASEAAYKEA (SEQ ID NO:32). Also provided arehybridoma cell lines capable of producing such antibodies.

In one aspect, the invention provides hybridomas produced by fusion of aspleen cell derived from a mouse immunized with an immunogen comprisinga 14-3-3 eta loop, helix, or non-helix peptide. In a preferredembodiment, the peptide comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs:1-32. In an especially preferredembodiment, the invention provides hybridomas produced by fusion ofspleen cells derived from mice immunized with an immunogen comprisingLDKFLIKNSNDF (SEQ ID NO:30), KKLEKVKAYR (SEQ ID NO:31), orKNSVVEASEAAYKEA (SEQ ID NO:32). Also provided are monoclonal antibodiesproduced by such hybridomas.

In a preferred embodiment, the invention provides monoclonal anti-14-3-3gamma antibodies that bind to a 14-3-3 gamma loop, helix, or non-helixpeptide. In a preferred embodiment, the peptide comprises an amino acidsequence selected from the group consisting of SEQ ID NOs:33-62. Alsoprovided are hybridoma cell lines capable of producing such antibodies.

In one aspect, the invention provides hybridomas produced by fusion of aspleen cell derived from a mouse immunized with an immunogen comprisinga 14-3-3 gamma loop, helix, or non-helix peptide. In a preferredembodiment, the peptide comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs:33-62. Also provided are monoclonalantibodies produced by such hybridomas.

The present disclosure further provides methods of producing suchmonoclonal antibodies, or derivatives thereof, comprising cultivating ahybridoma of the invention under suitable conditions, whereby amonoclonal antibody is produced, and obtaining the antibody and/orderivative thereof from the cell and/or from the cell culture medium.

Antibodies can be produced readily by one skilled in the art. Thegeneral methodology for making monoclonal antibodies by hybridomas isnow well known to the art. See, e.g., M. Schreier et al., HybridomaTechniques (Cold Spring Harbor Laboratory) 1980; Hammerling et al.,Monoclonal Antibodies and T-Cell Hybridomas (Elsevier Biomedical Press)1981.

In some embodiments, these methods comprise cultivating a hybridoma cellunder suitable conditions wherein the antibody is produced, andobtaining the antibody and/or derivative thereof from the cell and/orfrom the cell culture medium.

The present invention also contemplates the use of phage libraries topan for antibodies capable of binding to the 14-3-3 peptides of interestdescribed herein. For example, see Konthur et al., Targets, 1: 30-36,2002.

The antibodies produced by any means can be purified by methods known tothe skilled artisan. Purification methods include, among others,selective precipitation, liquid chromatography, HPLC, electrophoresis,chromatofocusing, and various affinity techniques. Selectiveprecipitation may use ammonium sulfate, ethanol (Cohn precipitation),polyethylene glycol, or other agents available in the art. Liquidchromatography mediums, include, among others, ion exchange medium DEAE,polyaspartate, hydroxylapatite, size exclusion (e.g., those based oncrosslinked agarose, acrylamide, dextran, etc.), hydrophobic matrices(e.g., Blue Sepharose). Affinity techniques typically rely on proteinsthat interact with the immunoglobulin Fc domain. Protein A fromStaphylococcus aureas can be used to purify antibodies that are based onhuman γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth.62:1-13 (1983)). Protein G from C and G streptococci is useful for allmouse isotypes and for human .γ3 (Guss et al., EMBO J. 5:15671575(1986)). Protein L, a Peptostreptococcus magnus cell-wall protein thatbinds immunoglobulins (Ig) through k light-chain interactions (BDBioscience/ClonTech. Palo Alto, Calif.), is useful for affinitypurification of Ig subclasses IgM, IgA, IgD, IgG, IgE and IgY.Recombinant forms of these proteins are also commercially available. Ifthe antibody contains metal binding residues, such as phage displayantibodies constructed to contain histidine tags, metal affinitychromatography may be used. When sufficient amounts of specific cellpopulations are available, antigen affinity matrices may be made withthe cells to provide an affinity method for purifying the antibodies.

In a preferred embodiment, isolation involves affinity chromatographyusing an appropriate 14-3-3 protein or fragment thereof.

The present invention provides the antibodies described herein, as wellas corresponding antibody fragments and antigen-binding portions. Allare encompassed by the term anti-14-3-3 antibody. The terms “antibodyfragment” or “antigen-binding portion” of an antibody (or simply“antibody portion”) of the present invention, as used herein, refers toone or more fragments of an antibody that retain the ability tospecifically bind to an antigen. It has been shown that theantigen-binding function of an antibody can be performed by fragments ofa full-length antibody. Examples of binding fragments encompassed withinthe term “antibody fragment” or “antigen-binding portion” of an antibodyinclude (i) a Fab fragment, a monovalent fragment consisting of the VL,VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) aFv fragment consisting of the VL and VH domains of a single arm of anantibody, (v) a dAb fragment (e.g., Ward et al., (1989) Nature341:544-546), which consists of a VH domain; and (vi) an isolatedcomplementarity determining region (CDR), and (vii) bispecific singlechain Fv dinners (e.g., PCT/US92/09965). Furthermore, although the twodomains of the Fv fragment, VL and VH, are coded for by separate genes,they can be joined, using recombinant methods, by a synthetic linkerthat enables them to be made as a single protein chain in which the VLand VH regions pair to form monovalent molecules (known as single chainFv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Hustonet al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such singlechain antibodies are also intended to be encompassed within the term“antigen-binding portion” of an antibody. These antibody fragments areobtained using conventional techniques known to those with skill in theart, and the fragments are screened for utility in the same manner asare intact antibodies. The antibody fragments may be modified. Forexample, the molecules may be stabilized by the incorporation ofdisulphide bridges linking the VH and VL domains (Reiter et al., 1996,Nature Biotech. 14:1239-1245).

Immunoglobulin molecules can be cleaved into fragments. The antigenbinding region of the molecule can be divided into either F(ab′)2 or Fabfragments. The F(ab′)2 fragment is divalent and is useful when the Fcregion is either undesirable or not a required feature. The Fab fragmentis univalent and is useful when an antibody has a very high avidity forits antigen. Eliminating the Fc region from the antibody decreasesnon-specific binding between the Fc region and Fc receptor bearingcells. To generate Fab or F(ab′)2 fragments, the antibodies are digestedwith an enzyme. Proteases that cleave at the hinge region of animmunoglobulin molecule preserve the disulfide bond(s) linking the Fabdomains such that they remain together following cleavage. A suitableprotease for this purpose is pepsin. For producing Fab fragments,proteases are chosen such that cleavage occurs above the hinge regioncontaining the disulfide bonds that join the heavy chains but whichleaves intact the disulfide bond linking the heavy and light chain. Asuitable protease for making Fab fragments is papain. The fragments arepurified by the methods described above, with the exception of affinitytechniques requiring the intact Fc region (e.g., Protein A affinitychromatography).

Antibody fragments can be produced by limited proteolysis of antibodiesand are called proteolytic antibody fragments. These include, but arenot limited to, the following: F(ab′)2 fragments, Fab′ fragments,Fab′-SH fragments, and Fab fragments. “F(ab′)2 fragments” are releasedfrom an antibody by limited exposure of the antibody to a proteolyticenzyme, e.g., pepsin or ficin. An F(ab′)2 fragment comprises two “arms,”each of which comprises a variable region that is directed to andspecifically binds a common antigen. The two Fab′ molecules are joinedby interchain disulfide bonds in the hinge regions of the heavy chains;the Fab′ molecules may be directed toward the same (bivalent) ordifferent (bispecific) epitopes. “Fab′ fragments” contain a singleantigen-binding domain comprising an Fab and an additional portion ofthe heavy chain through the hinge region. “Fab′-SH fragments” aretypically produced from F(ab′)2 fragments, which are held together bydisulfide bond(s) between the H chains in an F(ab′)2 fragment. Treatmentwith a mild reducing agent such as, by way of non-limiting example,beta-mercaptoethylamine, breaks the disulfide bond(s), and two Fab′fragments are released from one F(ab′)2 fragment. Fab′-SH fragments aremonovalent and monospecific. “Fab fragments” (i.e., an antibody fragmentthat contains the antigen-binding domain and comprises a light chain andpart of a heavy chain bridged by a disulfide bond) may be produced bypapain digestion of intact antibodies. A convenient method is to usepapain immobilized on a resin so that the enzyme can be easily removedand the digestion terminated. Fab fragments do not have the disulfidebond(s) between the H chains present in an F(ab′)2 fragment.

“Single-chain antibodies” are one type of antibody fragment. The termsingle chain antibody is often abbreviated as “scFv” or “sFv.” Theseantibody fragments are produced using recombinant DNA technology. Asingle-chain antibody consists of a polypeptide chain that comprisesboth a V_(H) and a V_(L) domains which interact to form anantigen-binding site. The V_(H) and V_(L) domains are usually linked bya peptide of 10 to 25 amino acid residues.

The term “single-chain antibody” further includes but is not limited toa disulfide-linked Fv (dsFv) in which two single-chain antibodies (eachof which may be directed to a different epitope) are linked together bya disulfide bond; a bispecific sFv in which two discrete scFvs ofdifferent specificity are connected with a peptide linker; a diabody (adimerized sFv formed when the V_(H) domain of a first sFv assembles withthe V_(L) domain of a second sFv and the V_(L) domain of the first sFvassembles with the V_(H) domain of the second sFv; the twoantigen-binding regions of the diabody may be directed towards the sameor different epitopes); and a triabody (a trimerized sFv, formed in amanner similar to a diabody, but in which three antigen-binding domainsare created in a single complex; the three antigen binding domains maybe directed towards the same or different epitopes).

“Complementary determining region peptides” or “CDR peptides” areanother form of an antibody fragment. In one embodiment, the inventionprovides such CDR peptides that are 14-3-3 antagonists. A CDR peptide(also known as “minimal recognition unit”) is a peptide corresponding toa single complementarity-determining region (CDR), and can be preparedby constructing genes encoding the CDR of an antibody of interest. Suchgenes are prepared, for example, by using the polymerase chain reactionto synthesize the variable region from RNA of antibody-producing cells.See, for example, Larrick et al., Methods: A Companion to Methods inEnzymology 2:106, 1991.

In “cysteine-modified antibodies,” a cysteine amino acid is inserted orsubstituted on the surface of antibody by genetic manipulation and usedto conjugate the antibody to another molecule via, e.g., a disulfidebridge. Cysteine substitutions or insertions for antibodies have beendescribed (see U.S. Pat. No. 5,219,996). Methods for introducing Cysresidues into the constant region of the IgG antibodies for use insite-specific conjugation of antibodies are described by Stimmel et al.(J. Biol. Chem 275:330445-30450, 2000).

The present disclosure further provides humanized and non-humanizedantibodies. Humanized forms of non-human (e.g., mouse) antibodies arechimeric antibodies that contain minimal sequence derived from non-humanimmunoglobulin. Generally, humanized antibodies are non-human antibodiesthat have had the variable-domain framework regions swapped forsequences found in human antibodies. The humanized antibodies may behuman immunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Generally, in a humanized antibody, the entire antibody, except theCDRs, is encoded by a polynucleotide of human origin or is identical tosuch an antibody except within its CDRs. The CDRs, some or all of whichare encoded by nucleic acids originating in a non-human organism, aregrafted into the beta-sheet framework of a human antibody variableregion to create an antibody, the specificity of which is determined bythe engrafted CDRs. The creation of such antibodies is described in,e.g., WO 92/11018, Jones, 1986, Nature 321:522-525, Verhoeyen et al.,1988, Science 239:1534-1536. Humanized antibodies can also be generatedusing mice with a genetically engineered immune system. e.g., Roque etal., 2004, Biotechnol. Prog. 20:639-654.

It can be desirable to modify the antibodies of the invention withrespect to effector function. For example, cysteine residue(s) can beintroduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. Homodimeric antibodies can also beprepared using heterobifunctional cross-linkers, e.g., Wolff et al.Cancer Research, 53:2560-2565 (1993). Alternatively, an antibody can beengineered that has dual Fc regions. See for example Stevenson et al.,Anti-Cancer Drug Design, 3:219-230 (1989).

Modified Antibodies

In one aspect, the present invention provides 14-3-3 antibodies that aremodified antibodies which are derived from an antibody that specificallybinds a 14-3-3 protein. Modified antibodies also include recombinantantibodies as described herein.

Numerous types of modified or recombinant antibodies will be appreciatedby those of skill in the art. Suitable types of modified or recombinantantibodies include, without limitation, engineered monoclonal antibodies(e.g. chimeric monoclonal antibodies, humanized monoclonal antibodies),domain antibodies (e.g. Fab, Fv, VH, scFV, and dsFv fragments),multivalent or multispecific antibodies (e.g. diabodies, minibodies,miniantibodies, (scFV)2, tribodies, and tetrabodies), and antibodyconjugates as described herein.

In one aspect, the present invention provides anti-14-3-3 antibodieswhich are domain antibodies. “Domain antibodies” are functional bindingdomains of antibodies, corresponding to the variable regions of eitherthe heavy (VH) or light (VL) chains of human antibodies. Domainantibodies may have a molecular weight of approximately 13 kDa, or lessthan one-tenth the size of a full antibody. They are well expressed in avariety of hosts including bacterial, yeast, and mammalian cell systems.In addition, domain antibodies are highly stable and retain activityeven after being subjected to harsh conditions, such as freeze-drying orheat denaturation. See, for example, U.S. Pat. Nos. 6,291,158;6,582,915; 6,593,081; 6,172,197; US Serial No. 2004/0110941; EuropeanPatent 0368684; U.S. Pat. No. 6,696,245, WO04/058821, WO04/003019 andWO03/002609. In one embodiment, the domain antibody of the presentinvention is a single domain. Single domain antibodies may be prepared,for example, as described in U.S. Pat. No. 6,248,516.

In another aspect, the present invention includes multi-specificantibodies. Multi-specific antibodies include bispecific, trispecific,etc. antibodies. Bispecific antibodies can be produced via recombinantmeans, for example by using leucine zipper moieties (i.e., from the Fosand Jun proteins, which preferentially form heterodimers; e.g., Kostelnyet al., 1992, J. Immnol. 148:1547) or other lock and key interactivedomain structures, for example as described in U.S. Pat. No. 5,582,996.Additional useful techniques include those described in U.S. Pat. Nos.5,959,083; and 5,807,706.

Bispecific antibodies are also sometimes referred to as “diabodies.”These are antibodies that bind to two (or more) different antigens. Alsoknown in the art are triabodies (a trimerized sFv, formed in a mannersimilar to a diabody, but in which three antigen-binding domains arecreated in a single complex; the three antigen binding domains may bedirected towards the same or different epitopes) or tetrabodies (fourantigen-binding domains created in a single complex where the fourantigen binding domains may be directed towards the same or differentepitopes). Dia-, tria- and tetrabodies can be manufactured in a varietyof ways known in the art (e.g., Holliger and Winter, 1993, CurrentOpinion Biotechnol. 4:446-449), e.g., prepared chemically or from hybridhybridomas. In addition, such antibodies and fragments thereof may beconstructed by gene fusion (e.g., Tomlinson et. al., 2000, MethodsEnzymol. 326:461-479; WO94/13804; Holliger et al., 1993, Proc. Natl.Acad. Sci. U.S.A. 90:6444-6448).

In another embodiment, the present invention provides minibodies, whichare minimized antibody-like proteins that include a scFV joined to a CH3domain, that are derived from an antibody that specifically binds 14-3-3protein. Minibodies can be made as described in the art (e.g., Hu etal., 1996, Cancer Res. 56:3055-3061).

In another embodiment, the present invention provides 14-3-3 bindingdomain-immunglobulin fusion proteins. In one embodiment, the fusionprotein may include a 14-3-3 binding domain polypeptide fused to animmunoglobulin hinge region polypeptide, which is fused to animmunoglobulin heavy chain CH2 constant region polypeptide fused to animmunoglobulin heavy chain CH3 constant region polypeptide. Under thepresent invention, 14-3-3 antibody fusion proteins can be made bymethods appreciated by those of skill in the art (See for examplepublished U.S. Patent Application Nos. 20050238646, 20050202534,20050202028, 2005020023, 2005020212, 200501866216, 20050180970, and20050175614).

In another embodiment, the present invention provides a heavy-chainprotein derived from a 14-3-3 antibody. Naturally-occurring heavy chainantibodies (e.g. camelidae antibodies having no light chains) have beenutilized to develop antibody-derived therapeutic proteins that typicallyretain the structure and functional properties of naturally-occurringheavy-chain antibodies. They are known in the art as Nanobodies. Heavychain proteins derived from a 14-3-3 heavy chain antibody may be made bymethods appreciated by those of skill in the art (See for examplepublished U.S. Patent Application Nos. 20060246477, 20060211088,20060149041, 20060115470, and 20050214857). Further, regarding theproduction of heavy chain-only antibodies in light chain-deficient mice,see for example Zou et al., JEM, 204:3271-3283, 2007.

In one aspect, the present invention provides a modified antibody thatis a human antibody. In one embodiment, fully human 14-3-3 antibodiesare provided. “Fully human antibody” or “complete human antibody” refersto a human antibody having only the gene sequence of an antibody derivedfrom a human chromosome. The anti-14-3-3 complete human antibody can beobtained by a method using a human antibody-producing mouse having ahuman chromosome fragment containing the genes for a heavy chain andlight chain of a human antibody [see for example Tomizuka, K. et al.,Nature Genetics, 16, p.133-143, 1997; Kuroiwa, Y. et al., Nuc. AcidsRes., 26, p. 3447-3448, 1998; Yoshida, H. et al., Animal CellTechnology: Basic and Applied Aspects vol. 10, p. 69-73 (Kitagawa, Y.,Matuda, T. and lijima, S. eds.), Kluwer Academic Publishers, 1999;Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA, 97, 722-727, 2000] orobtained by a method for obtaining a human antibody derived from a phagedisplay selected from a human antibody library (see for exampleWormstone, I. M. et al., Investigative Ophthalmology & Visual Science.43(7), p. 2301-8, 2002; Carmen, S. et al., Briefings in FunctionalGenomics and Proteomics, 1 (2), p. 189-203, 2002; Siriwardena, D. etal., Ophthalmology, 109(3), p. 427-431, 2002).

In one aspect, the present invention provides a 14-3-3 antibody that isan antibody analog, sometimes referred to as “synthetic antibodies.” Forexample, alternative protein scaffolds or artificial scaffolds withgrafted CDRs may be used. Such scaffolds include, but are not limitedto, synthetic scaffolds consisting, for example, of biocompatiblepolymers. See, for example, Korndorfer et al., 2003, Proteins:Structure, Function, and Bioinformatics, Volume 53, Issue 1:121-129.Roque et al., 2004, Biotechnol. Prog. 20:639-654. In addition, peptideantibody mimetics (“PAMs”) can be used, as well as antibody mimeticsutilizing fibronectin components as a scaffold.

In one aspect, the present invention provides cross-linked antibodiesthat include two or more antibodies described herein attached to eachother to form antibody complexes. Cross-linked antibodies are alsoreferred to as antibody multimers, homoconjugates, and heteroconjugates.

In some embodiments, the antibody complexes provided herein includemultimeric forms of anti-14-3-3 antibodies. For example, antibodycomplexes of the invention may take the form of antibody dimers,trimers, or higher-order multimers of monomeric immunoglobulinmolecules. Crosslinking of antibodies can be done through variousmethods know in the art. For example, crosslinking of antibodies may beaccomplished through natural aggregation of antibodies, through chemicalor recombinant linking techniques or other methods known in the art. Forexample, purified antibody preparations can spontaneously form proteinaggregates containing antibody homodimers, and other higher-orderantibody multimers.

In one embodiment, the present invention provides homodimerizedantibodies that specifically bind to 14-3-3 antigen.

Antibodies can be cross-linked or dimerized through linkage techniquesknown in the art. Non-covalent methods of attachment may be utilized. Ina specific embodiment, crosslinking of antibodies can be achievedthrough the use of a secondary crosslinker antibody. The crosslinkerantibody can be derived from a different animal compared to the antibodyof interest. For example, a goat anti-mouse antibody (Fab specific) maybe added to a mouse monoclonal antibody to form a heterodimer. Thisbivalent crosslinker antibody recognizes the Fab or Fc region of the twoantibodies of interest forming a homodimer.

In one embodiment of the present invention, an antibody thatspecifically binds to 14-3-3 antigen is cross-linked using a goatanti-mouse antibody (GAM). In another embodiment, the GAM crosslinkerrecognizes the Fab or Fc region of two antibodies, each of whichspecifically binds a 14-3-3 antigen.

Methods for covalent or chemical attachment of antibodies may also beutilized. Chemical crosslinkers can be homo or heterobifunctional andwill covalently bind with two antibodies forming a homodimer.Cross-linking agents are well known in the art; for example, homo-orhetero-bifunctional linkers as are well known (see the 2006 PierceChemical Company Crosslinking Reagents Technical Handbook; Hermanson, G.T., Bioconjugate Techniques, Academic Press, San Diego, Calif. (1996);Aslam M. and Dent A H., Bioconjugation: protein coupling techniques forthe biomedical sciences, Houndsmills, England: Macmillan Publishers(1999); Pierce: Applications Handbook & Catalog, Perbio Science,Ermbodegem, Belgium (2003-2004); Haughland, R. P., Handbook ofFluorescent Probes and Research Chemicals Eugene, 9th Ed., MolecularProbes, OR (2003); and U.S. Pat. No. 5,747,641) Those of skill in theart will appreciate the suitability of various functional groups on theamino acid(s) of an antibody for modification, including cross-linking.Suitable examples of chemical crosslinkers used for antibodycrosslinking include, but not limited to, SMCC [succinimidyl4-(maleimidomethyl)cyclohexane-1-carboxylate], SATA [N-succinimidylS-acethylthio-acetate], hemi-succinate esters of N-hydroxysuccinimide;sulfo-N-hydroxy-succinimide; hydroxybenzotriazole, and p-nitrophenol;dicyclohexylcarbodiimide (DCC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (ECD), and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodide (EDCI) (see,e.g., U.S. Pat. No. 4,526,714, the disclosure of which is fullyincorporated by reference herein). Other linking reagents includeglutathione, 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one(DEPBT), onium salt-based coupling reagents, polyoxyethylene-basedheterobifunctional cross-linking reagents, and other reagents (Haitao,et al., Organ Lett 1:91-94 (1999); Albericio et al., J Organic Chemistry63:9678-9683 (1998); Arpicco et al., Bioconjugate Chem. 8:327-337(1997); Frisch et al., Bioconjugate Chem. 7:180-186 (1996); Deguchi etal., Bioconjugate Chem. 10:32-37 (1998); Beyer et al., J. Med. Chem.41:2701-2708 (1998); Drouillat et al., J. Pharm. Sci. 87:25-30 (1998);Trimble et al., Bioconjugate Chem. 8:416-423 (1997)). An exemplaryprotocols for the formation of antibody homodimers is given in U.S.Patent Publication 20060062786. Techniques for conjugating therapeuticcompounds to antibodies are also described in Arnon et al., “MonoclonalAntibodies for Immunotargeting of Drugs in Cancers Therapy,” inMonoclonal Antibodies and Cancer Therapy, Reisfeld et al., ed., pp243-256, Alan R. Liss, Inc. (1985); Thorpe, et al. “The Preparation andCytotoxic Properties of Antibody Toxin Conjugates,” Immunol. Rev.62:119-58 (1982); and Pietersz, G. A., “The linkage of cytotoxic drugsto monoclonal antibodies for the treatment of cancer,” BioconjugateChemistry 1(2):89-95 (1990), all references incorporated herein byreference.

In addition, the antibody-antibody conjugates of this invention can becovalently bound to each other by techniques known in the art such asthe use of the heterobifunctional cross-linking reagents, GMBS(maleimidobutryloxy succinimide), and SPDP (N-succinimidyl3-(2-pyridyldithio)propionate) [see, e.g., Hardy, “Purification AndCoupling Of Fluorescent Proteins For Use In Flow Cytometry”, Handbook OfExperimental Immunology, Volume 1, Immunochemistry, Weir et al. (eds.),pp. 31.4-31.12 4th Ed., (1986), and Ledbetter et al. U.S. Pat. No.6,010,902].

In addition, antibodies may be linked via a thioether cross-link asdescribed in U.S. Patent Publication 20060216284, U.S. Pat. No.6,368,596. As will be appreciated by those skilled in the art,antibodies can be crosslinked at the Fab region. In some embodiments, itis desirable that the chemical crosslinker not interact with theantigen-binding region of the antibody as this may affect antibodyfunction.

Conjugated Antibodies

The 14-3-3 antagonists disclosed herein include antibodies conjugated toinorganic or organic compounds, including, by way of example and notlimitation, other proteins, nucleic acids, carbohydrates, steroids, andlipids (see for example Green, et al., Cancer Treatment Reviews,26:269-286 (2000). The compound may be bioactive. Bioactive refers to acompound having a physiological effect on the cell as compared to a cellnot exposed to the compound. A physiological effect is a change in abiological process, including, by way of example and not limitation, DNAreplication and repair, recombination, transcription, translation,secretion, membrane turnover, cell adhesion, signal transduction, celldeath, and the like. A bioactive compound includes pharmaceuticalcompounds. In one embodiment, a 14-3-3 antibody is conjugated to a14-3-3 antagonist peptide, preferably R-18, preferably via a linker.

Peptides

In one aspect, the invention provides 14-3-3 antagonists that arepeptides. Such peptides include CDR peptides.

In one embodiment, the peptide binds to a region of the 14-3-3 proteinthat is capable of binding to an anti-14-3-3 antibody or other 14-3-3antagonist peptide. The term “peptide” or “oligopeptide” as used hereinis meant to encompass peptide analogs, derivatives, fusion proteins andthe like, as well as peptide compositions, including those exemplifiedin the present disclosure.

In a preferred embodiment, the peptide comprises the amino acid sequencedesignated “R-18”. In another preferred embodiment, the peptide consistsessentially of the R-18 sequence. In another preferred embodiment, thepeptide comprises a segment of the R-18 sequence. In another preferredembodiment, the peptide comprises multiple iterations of the R-18sequence, preferably separated by a linker.

In another preferred embodiment, the peptide binds to a region of a14-3-3 protein that is capable of binding to R-18. In another preferredembodiment, the peptide binds to a region of a 14-3-3 protein that iscapable of binding to an intracellular 14-3-3 binding partner,preferably Raf.

In one embodiment, the peptide binds to a 14-3-3 protein withoutdisrupting binding of the 14-3-3 protein to an intracellular 14-3-3binding partner.

In one embodiment, the 14-3-3 antagonist is a phosphopeptide.

Peptide Modifications

The subject peptides may be modified in a variety of conventional wayswell known to the skilled artisan. Any number of modifications may bedone to achieve a peptide having desired characteristics. What isrequired of a peptide of the invention is that it retain the ability tofunction as a 14-3-3 antagonist.

Examples of modifications include the following. The terminal aminogroup and/or carboxyl group of the peptide and/or amino acid side chainsmay be modified by alkylation, amidation, or acylation to provideesters, amides or substituted amino groups. Heteroatoms may be includedin aliphatic modifying groups. This is done using conventional chemicalsynthetic methods. Other modifications include deamination of glutamyland asparaginyl residues to the corresponding glutamyl and aspartylresidues, respectively; hydroxylation of proline and lysine;phosphorylation of hydroxyl groups of serine or threonine; andmethylation of amino groups of lysine, arginine, and histidine sidechains (see, for example, T. E. Creighton, Proteins: Structure andMolecular Properties, W.H. Freeman & Co. San Francisco, Calif., 1983).

In another aspect, one or both, usually one terminus of the peptide, maybe substituted with a lipophilic group, usually aliphatic or aralkylgroup, which may include heteroatoms. Chains may be saturated orunsaturated. Conveniently, commercially available aliphatic fatty acids,alcohols and amines may be used, such as caprylic acid, capric acid,lauric acid, myristic acid and myristyl alcohol, palmitic acid,palmitoleic acid, stearic acid and stearyl amine, oleic acid, linoleicacid, docosahexaenoic acid, etc. (see U.S. Pat. No. 6,225,444).Preferred are unbranched, naturally occurring fatty acids between 14-22carbon atoms in length. Other lipophilic molecules include glyceryllipids and sterols, such as cholesterol. The lipophilic groups may bereacted with the appropriate functional group on the oligopeptide inaccordance with conventional methods, frequently during the synthesis ona support, depending on the site of attachment of the oligopeptide tothe support. Lipid attachment is useful where oligopeptides may beintroduced into the lumen of the liposome, along with other therapeuticagents for administering the peptides and agents into a host.

In additional embodiments, either or both the N- and C-terminus of thepeptide may be extended by not more than a total of about 100, usuallynot more than a total of about 30, more usually not more than about 20amino acids, often not more than about 9 amino acids, where the aminoacids will have fewer than 25%, more usually fewer than 20% polar aminoacids, more particularly, fewer than 20% which are charged amino acids.Thus, extensions of the above sequences in either direction are mainlydone with lipophilic, uncharged amino acids, particularly non-polaraliphatic amino acids and aromatic amino acids. The peptides maycomprise L-amino acids, D-amino acids, or mixtures of D- and L-aminoacids. Exceptions to the number of amino acid extensions arecontemplated when the oligopeptides are expressed as fusion or chimericproteins, as described below.

The peptides may also be in the form of oligomers, especially dimers ofthe peptides, which may be head to head, tail to tail, or head to tail,preferably with not more than about 6 repeats of the peptide. Theoligomer may contain one or more D-stereoisomer amino acids, up to allof the amino acids. The oligomers may or may not include linkersequences between the peptides. Suitable linkers include, but are notlimited to, those comprising uncharged amino acids and (Gly)n, where nis 1-7, Gly-Ser, Gly-Ala, Ala-Ser, or other flexible linkers, as knownin the art. Linkers of Gly or Gly-Ser may be used since these aminoacids are relatively unstructured, which allows interaction ofindividual peptides with cellular target molecules and limits structuralperturbations between peptides of the oligomer. It is to be understoodthat linkers other than amino acids may be used to construct theoligomeric peptides.

Peptides may also be in a structurally constrained form, such as cyclicpeptides, preferably of from about 9-50, usually 12 to 36 amino acids,where amino acids other than the specified amino acids may be present asa bridge. Thus, for example, addition of terminal cysteines allowsformation of disulfide bridges to form a ring peptide. In someinstances, one may use other than amino acids to cyclize the peptide.Bifunctional crosslinking agents are useful in linking two or more aminoacids of the peptide. Other methods for ring formation are known in theart, see for example Chen, S. et al., Proc. Natl. Acad. Sci. USA89:5872-5876 (1992); Wu, T. P. et al., Protein Engineering 6:471478(1993); Anwer, M. K. et al., Int. J. Pep. Protein Res. 36:392-399(1990); and Rivera-Baeza, C. et al. Neuropeptides 30: 327-333 (1996).Alternatively, structurally constrained peptides are made by addition ofdimerization sequences to the N- and C-terminal ends of the peptide,where interaction between dimerization sequences lead to formation of acyclic type structure (see, e.g., WO/0166565). In other instances, thesubject peptides are expressed as fusions to other proteins, whichprovide a scaffold for constrained display on a surface exposedstructure, such as a loop of a coiled-coil or β-turn structure.

Depending upon their intended use, particularly for administration tomammalian hosts, the subject peptides may also be modified by attachmentto other compounds for the purposes of incorporation into carriermolecules, changing peptide bioavailability, extending or shorteninghalf-life, controling distribution to various tissues or the bloodstream, diminishing or enhancing binding to blood components, and thelike. The subject peptides may be bound to these other components bylinkers which are cleavable or non-cleavable in the physiologicalenvironment, e.g., by MMPs in the synovium. The peptides may be joinedat any point of the peptide where a functional group is present, such ashydroxyl, thiol, carboxyl, amino, or the like. Desirably, modificationwill be at either the N-terminus or the C-terminus. For instance, thesubject peptides may be modified by covalently attaching polymers, suchas polyethylene glycol, polypropylene glycol, carboxymnethyl cellulose,dextran, polyvinyl alcohol, polyvinylpyrrolidine, polyproline,poly(divinyl-ether-co-malelc anhydride), poly(styrene-c-maleicanhydride), etc. Water-soluble polymers, such a polyethylene glycol andpolyvinylpyrrolidine are known to decrease clearance of attachedcompounds from the blood stream as compared to unmodified compounds. Themodifications can also increase solubility in aqueous media and reduceaggregation of the peptides. What is required is that the peptide, whendelivered and/or released, retains 14-3-3 antagonist function.

Peptide Conjugates and Fusion Proteins

In one embodiment, the peptide is conjugated to small molecules fordetection and isolation of the peptides, or to target or transport theoligopeptide to specific cells, tissues, or organs. Small moleculeconjugates include haptens, which are substances that do not initiate animmune response when introduced by themselves into an animal. Generally,haptens are small molecules of molecular weight less than about 2 kD,and more preferably less that about 1 kD. Haptens include small organicmolecules (e.g., p-nitrophenol, digoxin, heroin, cocaine, morphine,mescaline, lysergic acid, tetrahydrocannabinol, cannabinol, steroids,pentamidine, biotin, etc.). Binding to the hapten, for example forpurposes of detection or purification, are done with hapten specificantibodies or specific binding partners, such as avidin which bindsbiotin.

Small molecules that target the conjugate to specific cells or tissuesmay also be used.

It will be understood that labels well known in the art may also beattached to the peptides of the invention for use of such conjugates indiagnostic methods.

In one embodiment, the peptides are joined to any of a wide variety ofother peptides or proteins for a variety of purposes. The peptides maybe linked to peptides or proteins to provide convenient functionalitiesfor bonding, such as amino groups for amide or substituted amineformation, e.g., reductive amination; thiol groups for thioether ordisulfide formation; carboxyl groups for amide formation; and the like.Of particular interest are peptides of at least 2, more usually 3, andnot more than about 60 lysine groups, particularly polylysines of fromabout 4 to 20, usually 6 to 18 lysine units, referred to as multipleantigenic peptide system (MAPS), where the subject peptides are bondedto the lysine amino groups, generally at least about 20%, more usuallyat least about 50%, of available amino groups, to provide a multipeptideproduct (Butz, S. et al., Pept. Res. 7: 20-23 (1994)). In this way,molecules having a plurality of the subject peptides are obtained wherethe orientation of the subject peptides is in the same direction; ineffect, this linking group provides for tail-to-tail di- oroligomerization.

In one embodiment, the peptides are conjugated to other peptides orproteins for targeting the oligopeptide to cells and tissues, or addingadditional functionalities to the peptides. For targeting, the proteinor peptide used for conjugation will be selected based on the cell ortissue being targeted for therapy (Lee, R. et al., Arthritis. Rheum. 46:2109-2120 (2002); Pasqualini, R., Q. J. Nucl. Med. 43: 159-62 (1999);Pasgualinl, R., Nature 380: 364-366 (1996)). The proteins may alsocompromise poly-amino acids including, but not limited to, polyarginine;and polylysine, polyaspartic acid, etc., which may be incorporated intoother polymers, such as polyethylene glycol, for preparation of vesiclesor particles containing the conjugated peptides.

In one embodiment, the subject peptides may be expressed or synthesizedin conjunction with other peptides or proteins, to be a portion of thepolypeptide chain, either internal, or at the N-or C-terminus to formchimeric proteins or fusion proteins. By “fusion polypeptide” or “fusionprotein” or “chimeric protein” herein is meant a protein composed of aplurality of protein components that, while typically joined in thenative state, are joined by the respective amino and carboxy terminithrough a peptide linkage to form a continuous polypeptide. It will beappreciated that the protein components can be joined directly or joinedthrough a peptide linker/spacer.

Fusion polypeptides may be made to a variety of peptides or proteins todisplay the subject oligopeptides in a conformationally restricted form,for targeting to cells and tissues, for targeting to intracellularcompartments, tracking the fusion protein in a cell or an organism, andscreening for other molecules that bind the oligopeptides. Proteinsuseful for generating fusion proteins include various reporter proteins,structural proteins, cell surface receptors, receptor ligands, toxins,and enzymes. Exemplary proteins include fluorescent proteins (e.g.,Aequodia victoria GFP, Renilla renifornis GFP, Renilla muelledi GFP,luciferases, etc., and variants thereof); β-galactosidase; alkalinephosphatase; E. coli. maltose binding protein; coat proteins offilamentous bacteriophage; T cell receptor; charybdotoxin; and the like.

Fusion proteins also encompass fusions with fragments of proteins orother peptides, either alone or as part of a larger protein sequence.Thus, the fusion polypeptides may comprise fusion partners. By “fusionpartners” herein is meant a sequence that is associated with the peptidethat confers all members of the proteins in that class a common functionor ability. Fusion partners can be heterologous (i.e., not native to thehost cell) or synthetic (ie., not native to any cell). The fusionpartners include, but are not limited to, a) presentation structures,which provide the oligopeptides in a conformationally restricted orstable form; b) targeting sequences, which allow localization of thepeptide to a subcellular or extracellular compartment; c) stabilitysequences, which affects stability or protection from degradation to thepeptide or the nucleic acid encoding it; d) linker sequences, whichconformationally decouples the oligopeptide from the fusion partner; ande) any combination of the above.

In one aspect, the fusion partner is a presentation structure. By“presentation structure” as used herein is meant a sequence that whenfused to the subject peptides presents the peptides in aconformationally restricted form. Preferred presentation structuresenhance binding interactions with other binding partners by presenting apeptide on a solvent exposed exterior surface. Generally, suchpresentation structures comprise a first portion joined to theN-terminus of the oligopeptide and a second portion joined to theC-terminal end of the oligopeptide. That is, the peptide of the presentinvention is inserted into the presentation structures. Preferably, thepresentation structures are selected or designed to have minimalbiological activity when expressed in the target cells.

Preferably, the presentation structures maximize accessibility to thepeptides by displaying or presenting the peptide or an exterior loop.Suitable presentation structures include, but are not limited to, coiledcoil stem structures, minibody structures, loops on β-turns,dimerization sequences, cysteine linked structures, transglutaminaselinked structures, cyclic peptides, helical barrels, leucine zippermotifs, etc.

In one embodiment, the presentation structure is a coiled-coilstructure, which allows presentation of the subject peptide on anexterior loop (e.g., Myszka, D. G. et al., Biochemistry 33: 2363-2373(1994)), such as a coiled-coil leucine zipper domain (Martin, F. et al.,EMBO J. 13: 5303-5309 (1994)). The presentation structure may alsocomprise minibody structures, which is essentially comprised of aminimal antibody complementary region. The minibody structure generallyprovides two peptide regions that are presented along a single face ofthe tertiary structure in the folded protein (e.g., Bianchi, E. et al.,J. Mol. Biol. 236: 649-659 (1994); Tramontano, A. et al., J. Mol.Recognit. 7: 9-24 (1994)).

In another aspect, the presentation structure comprises two dimerizationsequences. The dimerization sequences, which can be same or different,associate non-covalently with sufficient affinity under physiologicalconditions to structurally constrain the displayed peptide. Thus, if adimerization sequence is used at each terminus of the subjectoligopeptide, the resulting structure can display the subject peptide ina structurally limited or constrained form. A variety of sequences aresuitable as dimerization sequences (see for example, WO 99/51625;incorporated by reference). Any number of protein-protein interactionsequences known in the art are useful for present purposes.

In a further aspect, the presentation sequence confers the ability tobind metal ions to generate a conformationally restricted secondarystructure. Thus, for example, C2H2 zinc finger sequences are used. C2H2sequences have two cysteines and two histidines placed such that a zincion is chelated. Zinc finger domains are known to occur independently inmultiple zinc-finger peptides to form structurally independent, flexiblylinked domains (e.g., Nakaseko, Y. et al., J. Mol. Biol. 228: 619-636(1992)). A general consensus sequence is (5 amino acids)-C-(2 to 3 aminoacids)-C-(4 to 12 amino acids)-H-(3 amino acids)-H-(5 amino acids) (SEQID NO:66). A preferred example would be -FQCEEC-random peptide of 3 to20 amino acids-HIRSHTG (SEQ ID NO:67). Similarly, CCHC boxes having aconsensus sequence -C-(2 amino acids)-C-(4 to 20 random peptide)-H-(4amino acids)-C- (SEQ ID NO:68) can be used, (Bavoso, A. et al., Biochem.Biophys. Res. Commun. 242: 385389 (1998)). Other examples include(1)-VKCFNC-4 to 20 random amino acids-HTARNCR- (SEQ ID NO: 69), based onthe nucleocapsid protein P2; (2) a sequence modified from that of thenaturally occurring zinc-binding peptide of the Lasp-1 LIM domain(Hammarstrom, A. et al., Biochemistry 35: 12723-32 (1996)); and (3)-MNPNCARCG-4 to 20 (Hammarstrom et al., supra).

In yet another aspect, the presentation structure is a sequence thatcomprises two or more cysteine residues, such that a disulfide bond maybe formed, resulting in a conformationally constrained structure. Thatis, use of cysteine containing peptide sequences at each terminus of thesubject oligopeptides results in cyclic peptide structures, as describedabove. A cyclic structure reduces susceptibility of the presentedpeptide to proteolysis and increases accessibility to its targetmolecules. As will be appreciated by those skilled in the art, thisparticular embodiment is particularly suited when secretory targetingsequences are used to direct the peptide to the extracellular space. Inaddition, sequences that are recognized and cleaved by proteases, suchas the matrix metalloproteases (e.g., MMP-1, MMP-3), may be used. Theseresidues are used to form circular peptides to increase peptidehalf-life or membrane permeability. Subsequent cleavage of the circularpeptide with the appropriate protease releases the active, linear formof the peptide at the desired location.

In another embodiment, the fusion partner is a targeting sequence.Targeting sequences comprise binding sequences capable of causingbinding of the expressed product to a predetermined molecule or class ofmolecules while retaining bioactivity of the expression product;sequences signaling selective degradation of the fusion protein orbinding partners; and sequences capable of constitutively localizingpeptides to a predetermined cellular locale. Typical cellular locationsinclude subcellular locations (e.g., Golgi, endoplasmic recticulum,nucleus, nucleoli, nuclear membrane, mitochondria, secretory vesicles,lysosomes) and extracellular locations by use of secretory signals.

Various targeting sequences are known in the art, includingmembrane-anchoring sequences. Peptides are directed to the membrane viasignal sequences and stably incorporated in the membrane through ahydrophobic transmembrane domain (designated as TM). The TM segment ispositioned appropriately on the expressed fusion protein to display thesubject peptide either intracellularly or extracellularly, as is knownin the art. Especially preferred is extracellular presentation. Membraneanchoring sequences and signal sequences include, but are not limitedto, those derived from (a) class I integral membrane proteins such asIL-2 receptor β-chain; Hatakeyama, M. et al., Science 244: 551-556(1989)) and insulin receptor β-chain (Hetakeyama et al, supra); (b)class II integral membrane proteins such as neutral endopeptidase(Malfroy, B. et al Biochem. Biophys. Res. Commun. 144: 59-66 (1987));and (c) type III proteins such as human cytochrome P450 NF25 (Hetakeyamaet al, supra); and those from CD8, ICAM-2, IL-8R, and LFA-1.

Membrane anchoring sequences also include the GPI anchor, which resultsin covalent bond formation between the GPI anchor sequence and the lipidbilayer via a glycosyl-phosphatidylinositol. GPI anchor sequences arefound in various proteins, including Thy-1 and DAF (Homans, S. W. etal., Nature 333: 269-272 (1988)). Similarly, acylation sequences allowfor attachment of lipid moieties, e.g., isoprenylation (ie., famesyl andgeranyl-geranyl; see Farnsworth, C. C. et al., Proc. Natl. Aced. Sci.USA 91: 11963-11967 (1994) and Aronheim, A. et al., Cell 78: 949-61(1994)), myristoylation (Stickney, J. T. Methods Enzymol. 332: 64-77(2001)), or palmitoylation. In one aspect, the subject peptide will bebound to a lipid group at a terminus, so as to be able to be bound to alipid membrane, such as a liposome.

In another aspect, the targeting sequence is a secretory signal sequencewhich effects secretion of the peptide. A large number of secretorysequences are known to direct secretion of a peptide into theextracellular space when placed at the amino end relative to the peptideof interest, particularly for secretion of a peptide by cells, includingtransplanted cells. Suitable secretory signals included those found inIL-2 (Villinger, F. et al., J. Immuno. 155: 3946-3954 (1995)), growthhormone (Roskam, W. G. et al., Nucleic Acids Res. 7: 305-320 (1979)),preproinsulin, and influenza HA protein.

The fusion partner may further comprise a stability sequence, whichconfers stability to the fusion protein or the nucleic acid encoding it.Thus, for example, incorporation of glycines after the initiatingmethionine (e.g., MG or MGG) can stabilize or protect the fused peptidefrom degradation via ubiquitination as per the N-End rule of Varshaysky,thus conferring increased half-life in a cell.

Additional amino acids may be added for tagging the peptide for purposesof detection or purification. These sequences may comprise epitopesrecognized by antibodies or sequences that bind ligands, such a metalsions. Various tag sequences and ligand binding sequences are well knownin the art. These include, but is not limited to, poly-histidine (e.g.,6×His tags, which are recognized by antibodies but also bind divalentmetal ions); poly-histidine-glycine (poly-his-gly) tags; flu HA tagpolypeptide; c-myc tag; Flag peptide (e.g., Hopp et al., BioTechnology6: 1204-1210 (1988)); KT3 epitope peptide; tubulin epitope peptide(e.g., Skinner et al., J. Biol. Chem. 266: 15163-12166 (1991)); and T7gene 10 protein peptide tag (e.g., Lutz-Freyermuth et al., Proc. Natl.Acad. Sci. USA 87: 6363-6397 (1990)).

Fusion partners include linker or tethering sequences, as discussedherein, for linking the peptides and for presenting the peptides in anunhindered structure.

In the present invention, combinations of fusion partners may be used.Any number of combinations of presentation structures, targetingsequences, tag sequences and stability sequences may be used with orwithout linker sequences.

Peptide Preparation and Salts

The peptides of the invention may be prepared in a number of ways.Chemical synthesis of peptides is well known in the art. Solid phasesynthesis is commonly used and various commercial synthetic apparatusesare available, for example automated synthesizers by Applied BiosystemsInc., Foster City, Calif.; Beckman; etc. Solution phase syntheticmethods may also be used, particularly for large-scale productions. Byusing these standard techniques, naturally occurring amino acids may besubstituted with unnatural amino acids, particularly D-stereoisomers,and with amino acids with side chains having different lengths orfunctionalities. Functional groups for conjugating to small molecules,label moieties, peptides, or proteins, or for purposes of formingcyclized peptides may be introduced into the molecule during chemicalsynthesis. In addition, small molecules and label moieties may beattached during the synthetic process. Preferably, introduction of thefunctional groups and conjugation to other molecules minimally affectsthe structure and function of the subject peptide.

The peptides of the present invention may also be present in the form ofa salt, generally in a salt form which is pharmaceutically acceptable.These include inorganic salts of sodium, potassium, lithium, ammonium,calcium, magnesium, iron, zinc, copper, manganese, and the like.

Various organic salts of the peptide may also be made with, including,but not limited to, acetic acid, propionic acid, pyruvic acid, maleicacid, succinic acid, tartaric acid, citric acid, benozic acid, cinnamicacid, salicylic acid, etc.

Synthesis of the oligopeptides and derivatives thereof may also becarried out by using recombinant techniques. For recombinant production,a nucleic acid sequence may be made which encodes a single oligopeptideor preferably a plurality of the subject peptides in tandem with anintervening amino acid or sequence, which allows for cleavage to thesingle peptide or head to tail dimers. Where methionine or tryptophaneis absent, an intervening methionine or tryptophane may be incorporated,which allows for single amino acid cleavage using CNBr or BNPS-Skatole(2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine), respectively.Alternatively, cleavage is accomplished by use of sequences that arerecognized by particular proteases for enzymatic cleavage or sequencesthat act as self-cleaving sites (e.g., 2A sequences of apthoviruses andcardioviruses; Donnelly, M. L., J. Gen. Virol. 78:13-21 (1997);Donnelly, M. L., J. Gen. Virol. 82:1027-41 (2001)). The subject peptidemay also be made as part of a larger peptide, which can be isolated andthe oligopeptide obtained by proteolytic cleavage or chemical cleavage.The particular sequence and the manner of preparation will be determinedby convenience, economics, purity required, and the like. To preparethese compositions, a gene encoding a particular peptide, protein, orfusion protein is joined to a DNA sequence encoding the oligopeptides ofthe present invention to form a fusion nucleic acid, which is introducedinto an expression vector. Expression of the fusion nucleic acid isunder the control of a suitable promoter and other control sequences, asdefined below, for expression in a particular host cell or organism(Sambrook et al., Molecular Biology: A Laboratory Manual, Cold SpringHarbor Press, Cold Spring Harbor, N.Y. (3rd ed. 2001); Ausubel, F. etal., Current Protocols in Molecular Biology, John Wiley & Sons, NewYork, N.Y., (updates up to 2002) (1988)).

For conjugating various molecules to the peptides of the presentinvention, functional groups on the oligopeptides and the other moleculeare reacted in presence of an appropriate conjugating (e.g.,crosslinking) agent. The type of conjugating or crosslinking agent usedwill depend on the functional groups, such as primary amines,sulfhydryls, carbonyls, carbohydrates and carboxylic acids being used.Preferably, reactive functional groups on the oligopeptide not selectedfor modification are protected prior to coupling of the peptide to otherreactive molecules to limit undesired side reactions. By “protectinggroup” as used herein is a molecule bound to a specific functional groupwhich is selectively removable to reexpose the functional group (Greene,T. W. and Wuts, P. G. M. Protective Groups in Organic Synthesis, JohnWiley & Sons, Inc., New York (3^(ed.) 1999)). The peptides may besynthesized with protected amino acid precursors or reacted withprotecting groups following synthesis but before reacting withcrosslinking agent. Conjugations may also be indirect, for example byattaching a biotin moiety, which can be contacted with a compound ormolecule which is coupled to streptavidin or avidin.

For oligopeptides that have reduced activity in the conjugated form, ina preferred embodiment, the linkage between the oliogopeptides and theconjugated compound is chosen to be sufficiently labile to result incleavage under desired conditions, for example after transport todesired cells or tissues. Biologically labile covalent bonds, e.g.,lmimo bonds and esters, are well known in the art (see, e.g., U.S. Pat.No. 5,108,921). These modifications permit administration of theoligopeptides in potentially a less active form, which is then activatedby cleavage of the labile bond.

Nucleic Acids, Expression Vectors, and Methods of Introduction

14-3-3 antagonists which are proteins, including peptides andantibodies, may be synthesized using nucleic acids encoding the same.This may be done to produce 14-3-3 antagonists which are subsequentlyisolated for use. Alternatively, such nucleic acids may be usedtherapeutically.

In one embodiment, the nucleic acids are cloned into expression vectorsand introduced into cells or a host. The expression vectors are eitherself-replicating extrachromosomal vectors or vectors that integrate intothe host chromosome, for example vectors based on retroviruses, vectorswith site specific recombination sequences, or by homologousrecombination. Generally, these vectors include control sequencesoperably linked to the nucleic acids encoding the oligopeptides. By“control sequences” is meant nucleic acid sequences necessary forexpression of the subject peptides in a particular host organism. Thus,control sequences include sequences required for transcription andtranslation of the nucleic acids, including, but not limited to,promoter sequences, enhancer or transcriptional activator sequences,ribosomal binding sites, transcriptional start and stop sequences;polyadenylation signals; etc.

In a preferred embodiment, the cell is a fibroblast or FLS cell.Preferably the cells is a synovial cell. Preferably the cell isengineered to express a 14-3-3 antagonist. The cell may be manipulatedin vitro and introduced into a recipient. Alternatively, the cell may bemanipulated in vivo.

A variety of promoters are useful in expressing the peptides of thepresent invention. The promoters may be constitutive, inducible, and/orcell specific, and may comprise natural promoters, synthetic promoters(e.g., tTA tetracycline inducible promoters), or hybrids of variouspromoters. Promoters are chosen based on, among other considerations,the cell or organism in which the proteins are to be expressed, thelevel of desired expression, and any desired regulation of expression.Suitable promoters are bacterial promoters (e.g., pL1 phage promoter,tac promoter, lac promoter, etc.); yeast based promoters (e.g., GAL4promoter, alcohol dehydrogenase promoter, tryptophane synthase promoter,copper inducible CUPI promoter, etc.), plant promoters (e.g., CaMV S35,nopoline synthase promoter, tobacco mosaic virus promoter, etc), insectpromoters (e.g., Autographa nuclear polyhedrosis virus, Aedes DNV viralp& and p61, hsp70, etc.), and promoters for expression mammalian cells(e.g., ubiquitin gene promoter, ribosomal gene promoter, β-globinpromoter, thymidine kinase promoter, heat shock protein promoters, andribosomal gene promoters, etc.), and particularly viral promoters, suchas cytomegalovirus (CMV) promoter, simian virus (SV40) promoter, andretroviral promoters.

By “operably linked” herein is meant that a nucleic acid is placed intoa functional relationship with another nucleic acid. In the presentcontext, operably linked means that the control sequences are positionedrelative to the nucleic acid sequence encoding the subject 14-3-3antagonists in such a manner that expression of the encoded antagonistoccurs. The vectors may comprise plasmids or comprise viral vectors, forexample retroviral vectors, which are useful delivery systems if thecells are dividing cells, or lentiviral and adenoviral vectors if thecells are non-dividing cells. Particularly preferred areself-inactivating retroviral vectors (SIN vectors), which haveinactivated viral promoters at the 3′-LTR, thereby permitting control ofexpression of heterologous genes by use of non-viral promoters insertedinto the viral vector (see, e.g., Hofmann, A. et al., Proc. Natl. Acad.Sci. USA 93: 5185-5190 (1996)). As will be appreciated by those in theart, modifications of the system by pseudotyping allows use ofretroviral vectors for all eukaryotic cells, particularly for highereukaryotes (Morgan, R. A. et al., J. Virol. 67: 4712-4721 (1993); Yang,Y. et al., Hum. Gene Ther. 6:1203-1213 (1995)).

In addition, the expression vectors also contain a selectable markergene to allow selection of transformed host cells. Generally, theselection will confer a detectable phenotype that enriches for cellscontaining the expression vector and further permits differentiationbetween cells that express and do not express the selection gene.Selection genes are well known in the art and will vary with the hostcell used. Suitable selection genes include genes that render the cellresistant to a drug, genes that permit growth in nutritionally deficientmedia, and reporter genes (e.g., β-galactosidase, fluorescent proteins,glucouronidase, etc.), all of which are well known in the art andavailable to the skilled artisan.

There are a variety of techniques available for introducing nucleicacids into viable cells. By “introduced” into herein is meant that thenucleic acid enters the cells in a manner suitable for subsequentexpression of the nucleic acid. Techniques for introducing the nucleicacids will vary depending on whether the nucleic acid is transferred invitro into cultured cells or in vivo into the cells of the intended hostorganism and will also depend on the type of host organism. Methods forintroducing the nucleic acids in vitro include the use of liposomes,Lipofectin™, electroporation, microinjection, cell fusion, DEAE dextran,calcium phosphate precipitation, and biolistic particle bombardment.Techniques for transfer in vivo include direct introduction of thenucleic acid, use of viral vectors, typically retroviral vectors, andliposome mediated transfection, including viral coated liposome mediatedtransfection. The nucleic acids expressing the 14-3-3 antagonists of thepresent invention may exist transiently or stably in the cell or stablyintegrate into the chromosome of the host.

In some situations, it is desirable to include an agent that targets thetarget cells or tissues, such as an antibody specific for a cell surfaceprotein or the target cell e.g., a fibroblast or FLS cell, a ligand fora receptor on the target cell, a lipid component on the cell membrane,or a carbohydrate on the cell surface. If liposomes are employed,proteins that bind a cell surface protein which is endocytosed may beused for targeting and/or facilitating uptake. These include asnon-limiting examples, capsid proteins or fragments thereof tropic for aparticular cell types, antibodies for proteins which undergointernalization (Wu, G. Y. et al., J. Biol. Chem. 262: 4429-4432 (1987);Wagner, E. et al., Proc. Natl. Aced. Sci. USA 87: 3410-3414 (1990)), orenhance in vivo half-life.

Expression is done in a wide range of host cells that span prokaryotesand eukaryotes, including bacteria, yeast, plants, insects, and animals.The oligopeptides of the present invention may be expressed in, amongothers, E. coli, Saccharomyces cerevisae, Saccharomyces pombe, Tobaccoor Arabidopsis plants, insect Schneider cells, and mammalian cells, suchas COS, CHO, HeLa, and the like, either intracellularly or in a secretedform by fusing the peptides to an appropriate signal peptide. Secretionfrom the host cell may be done by fusing the DNA encoding theoligopeptide and a DNA encoding a signal peptide. Secretory signals arewell known in the art for bacteria, yeast, insects, plants, andmammalian systems. Nucleic acids expressing the oligopeptides may beinserted into cells, for example stem cells for tissue expression orbacteria for gut expression, and the cells transplanted into the host toprovide an in vivo source of the oligopeptides.

Purified Peptides

In a preferred embodiment, the oligopeptides of the present inventionmay be purified or isolated after synthesis or expression. By “purified”or “isolated” is meant free from the environment in which the peptide issynthesized or expressed and in a form where it can be practically used.Thus, by purified or isolated is meant that the peptide or itsderivative is substantially pure, i.e., more than 90% pure, preferablymore than 95% pure, and preferably more than 99% pure. The oligopeptidesand derivatives thereof may be purified and isolated by methods known tothose skilled in the art, depending on other components present in thesample. Standard purification methods include electrophoretic,immunological, and chromatographic techniques, including ion exchange,hydrophobic, affinity, size exclusion, reverse phase HPLC, andchromatofocusing. The proteins may also be purified by selectivesolubility, for instance in the presence of salts or organic solvents.The degree of purification necessary will vary depending on use of thesubject oligopeptides. Thus, in some instances no purification will benecessary.

For most applications, the compositions used will comprise at least 20%by weight of the desired product, more usually at least about 75% byweight, preferably at least about 95% by weight, and usually at leastabout 99.5% by weight, relative to contaminants related to the method ofproduct preparation, the purification procedure, and its intended use,for example with a pharmaceutical carrier for the purposes oftherapeutic treatment. Usually, the percentages will be based upon totalprotein.

Pharmaceutical Compositions, Administration, and Dosages

The 14-3-3 antagonists of the invention can be incorporated intopharmaceutical compositions suitable for administration to a subject.Typically, the pharmaceutical composition comprises a 14-3-3 antagonistof the invention and a pharmaceutically acceptable carrier. As usedherein, “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Examples of pharmaceutically acceptablecarriers include one or more of water, saline, phosphate bufferedsaline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In many cases, it will be preferable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Pharmaceuticallyacceptable substances such as wetting or minor amounts of auxiliarysubstances such as wetting or emulsifying agents, preservatives orbuffers, which enhance the shelf life or effectiveness of the 14-3-3antagonist.

The 14-3-3 antagonists are targeted to 14-3-3 protein that is localizedextracellularly. Accordingly, therapeutic compositions are formulatedand administration is such that the 14-3-3 antagonist so delivered isavailable to engage extracellular 14-3-3 protein.

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The preferred form depends on the intended mode of administration andtherapeutic application. Typical preferred compositions are in the formof injectable or infusible solutions, such as compositions similar tothose used for passive immunization of humans with other antibodies. Thepreferred mode of administration is parenteral (e.g., intravenous,subcutaneous, intraperitoneal, intramuscular, with intracapsular beingespecially preferred). In one embodiment, the 14-3-3 antagonist isadministered by intravenous infusion or injection. In another preferredembodiment, the 14-3-3 antagonist is administered by intramuscular orsubcutaneous injection. In a preferred embodiment, direct injection intothe synovium is done.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the active compound in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The proper fluidity of a solution can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

The 14-3-3 antagonists of the present invention can be administered by avariety of methods known in the art, including intravenous injection orinfusion. Direct administration to the synovium is one preferred routeof administration. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults. In certain embodiments, the active compound may be preparedwith a carrier that will protect the compound against rapid release,such as a controlled release formulation, including implants,transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.Representative formulation technology is taught in, inter alia,Remington: The Science and Practice of Pharmacy, 19th Ed., MackPublishing Co., Easton, Pa. (1995) and Handbook of PharmaceuticalExcipients, 3rd Ed, Kibbe, A. H. ed., Washington D.C., AmericanPharmaceutical Association (2000)

In certain embodiments, a 14-3-3 antagonist of the invention may beorally administered, for example, with an inert diluent or anassimilable edible carrier. The compound (and other ingredients, ifdesired) may also be enclosed in a hard or soft shell gelatin capsule,compressed into tablets, or incorporated directly into the subject'sdiet. For oral therapeutic administration, the compounds may beincorporated with excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. To administer a compound of the invention by other thanparenteral administration, it may be necessary to coat the compoundwith, or co-administer the compound with, a material to prevent itsinactivation.

Supplementary active compounds can also be incorporated into thecompositions. In certain embodiments, a 14-3-3 antagonist of theinvention is coformulated with and/or coadministered with one or moreadditional therapeutic agents. For example, a DMARD or DMOAD. Suchcombination therapies may advantageously utilize lower dosages of theadministered therapeutic agents, thus avoiding possible toxicities orcomplications associated with the various monotherapies.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an antibody of the invention. A “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired therapeutic result. Atherapeutically effective amount of the 14-3-3 antagonist may varyaccording to factors such as the disease state, age, sex, and weight ofthe individual, and the ability of the 14-3-3 antagonist to elicit adesired response in the individual. A therapeutically effective amountis also one in which any toxic or detrimental effects of the antibodyare outweighed by the therapeutically beneficial effects. A“prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeutic orprophylactic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody of the invention is0.1-20 mg/kg, more preferably 1-10 mg/kg. It is to be noted that dosagevalues may vary with the type and severity of the condition to bealleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.

The pharmaceutical compositions described herein may be presented inunit-dose or multi-dose containers, such as sealed ampoules or vials.Such containers are typically sealed in such a way to preserve thesterility and stability of the formulation until use. In general,formulations may be stored as suspensions, solutions or emulsions inoily or aqueous vehicles, as indicated above. Alternatively, apharmaceutical composition may be stored in a freeze-dried conditionrequiring only the addition of a sterile liquid carrier immediatelyprior to use.

Therapeutic Use of 14-3-3 Antagonists

By “treatment” herein is meant therapeutic or prophylactic treatment, ora suppressive measure for the disease, disorder or undesirablecondition. Treatment encompasses administration of the subject 14-3-3antagonists in an appropriate form prior to the onset of diseasesymptoms and/or after clinical manifestations, or other manifestations,of the disease to reduce disease severity, halt disease progression, oreliminate the disease. Prevention of the disease includes prolonging ordelaying the onset of symptoms of the disorder or disease, preferably ina subject with increased susceptibility to the disease.

In one aspect, the invention provides methods of treating arthritis,including methods of treating ankylosing spondylitis, Behçet's Disease,diffuse idiopathic skeletal hyperostosis (DISH), Ehlers-Danlos Syndrome(EDS), Felty's Syndrome, fibromyalgia, gout, infectious arthritis,juvenile arthritis, lupus, mixed connective tissue disease (MCTD),osteoarthritis, Paget's Disease, polymyalgia rheumatica, polymyositisand dermatomyositis, pseudogout, psoriatic arthritis, Raynaud'sPhenomenon, reactive arthritis, rheumatoid arthritis, scleroderma,Sjögren's Syndrome, Still's Disease, and Wegener's granulomatosis.

Generally, the methods comprise administering a 14-3-3 antagonist to apatient, either alone or in combination with other therapeutic agents toincrease treatment efficacy.

Methods of Screening for 14-3-3 Antagonists

In one aspect, the invention provides methods of screening for 14-3-3antagonists. The compounds screened can range from small organicmolecules to large polymers and biopolymers, and can include, by way ofexample and not limitation, small organic compounds, saccharides,carbohydrates, polysaccharides, lectins, peptides and analogs thereof,polypeptides, proteins, antibodies, oligonucleotides, polynucleotides,nucleic acids, etc.

In one embodiment, the candidate compounds screened are small organicmolecules, preferably having a molecular weight in the range of about100-2500 daltons, though other molecules may be used. Such candidatemolecules will often comprise cyclical structures composed of carbonatoms or mixtures of carbon atoms and one or more heteroatoms and/oraromatic, polyaromatic, heteroaromatic and/or polyaromatic structures.The candidate agents may include a wide variety of functional groupsubstituents. In one embodiment, the substituent(s) are independentlyselected from the group of substituents known to interact with proteins,such as, for example, amine, carbonyl, hydroxyl and carboxyl groups.

The candidate compounds may be screened on a compound-by-compound basisor, alternatively, using one of the myriad library techniques commonlyemployed in the art. For example, synthetic combinatorial compoundlibraries, natural products libraries and/or peptide libraries may bescreened using the assays of the invention to identify compounds thatcompete with a 14-3-3 ligand for binding to a 14-3-3 protein. Thesecompetitive binding assays can identify compounds that bind the 14-3-3protein at approximately the same site as the 14-3-3 ligand. Myriadtechniques for carrying out competitive binding assays are known in theart. Any of these techniques may be employed in the present invention.

Such binding experiments may be conducted wholly in solution or,alternatively, using a solid support, e.g., a glass or other bead, or asolid surface such as, for example, the bottom of a petri dish, toimmobilize a reagent. The immobilization may be mediated by non-covalentinteractions or by covalent interactions. Methods for immobilizingmyriad types of compounds and proteins on solid supports are well-known.Any of these methods may be used.

Whether carried out in solution or with an immobilized 14-3-3 protein orcandidate compound, the 14-3-3 protein and candidate compound aretypically contacted with one another under conditions conducive tobinding. Although the actual conditions used can vary, typically thebinding assays are carried out under physiological conditions. Actualconcentrations suitable for a particular assay will be apparent to thoseof skill in the art.

In one embodiment, the assays further comprise functional assays for theability of a candidate agent to antagonize 14-3-3 protein activity. Inone embodiment, the assays comprise determining the ability of acandidate agent to reduce the induction of MMP by 14-3-3 protein. In oneembodiment, candidate agent is mixed with 14-3-3 protein, and themixture may be added to cells capable of inducing MMP in response to14-3-3 protein. In another embodiment, candidate agent is added with14-3-3 protein to cells capable of inducing MMP in response to 14-3-3protein. Other functional assays measuring the ability of a candidateagent to inhibit the ability of a 14-3-3 protein to induce a measurablechange in cells, preferably fibroblasts or FLS cells, and thus tocharacterize the candidate as a 14-3-3 antagonist, can be undertaken.

All citations herein, including those listed below, are expresslyincorporated herein in their entirety by reference.

EXPERIMENTAL

TABLE 1 14-3-3 Eta epitopes SEQ ID NO: 1  93-107 helix LETVCNDVLSLLDKFSEQ ID NO: 2 191-199 helix EQACLLAKQ SEQ ID NO: 3 144-155 helixNSVVEASEAAYK SEQ ID NO: 4 144-152 helix NSVVEASEA SEQ ID NO: 5 147-155helix VEASEAAYK SEQ ID NO: 6 163-170 helix EQMQPTHP SEQ ID NO: 7 168-177helix THPIRLGLAL SEQ ID NO: 8  82-92 helix VKAYTEKIEKE SEQ ID NO: 9 68-79 helix QKTMADGNEKKL SEQ ID NO: 10 138-146 helix ASGEKKNSVSEQ ID NO: 11  69-77 loop KTMADGNEK SEQ ID NO: 12  32-40 loop ELNEPLSNESEQ ID NO: 13 103-117 loop LLDKFLIKNCNDFQY SEQ ID NO: 14 130-143 loopYYRYLAEVASGEKK SEQ ID NO: 15 184-194 loop YEIQNAPEQAC SEQ ID NO: 16206-218 loop AELDTLNEDSYKD SEQ ID NO: 17  44-57 non-helix LLSVAYKNVVGARRSEQ ID NO: 18  15-23 non-helix EQAERYDDM SEQ ID NO: 19 130-138 non-helixYYRYLAEVA SEQ ID NO: 20 118-125 non-helix ESKVFYLK SEQ ID NO: 21 210-218non-helix TLNEDSYKD SEQ ID NO: 22  77-84 non-helix KKLEKVKASEQ ID NO: 23  76-86 non-helix EKKLRKVKAYR SEQ ID NO: 24 142-158non-helix KKNSVVEASEAAYKE AF SEQ ID NO: 25 105-120 non-helixDKFLIKNCNDFQYESK SEQ ID NO: 26 237-246 non-helix QQDEEAGEGNSEQ ID NO: 27  75-82 non-helix NEKKLEKVK SEQ ID NO: 28 104-116 non-helixLDKFLIKNCNDFQ SEQ ID NO: 29 141-146 non-helix EKKNSV SEQ ID NO: 30104-115 non-helix LDKFLIKNS*NDF SEQ ID NO: 31  77-86 non-helixKKLEKVKAYR SEQ ID NO: 32 143-157 non-helix KNSVVEASEAAYKEA SEQ ID NO: 65  1-12 non-helix DREQLLQRARLA *The internal cysteine amino acid wasreplaced by the amino acid serine to prevent formation of disulfidebonds.

TABLE 2 14-3-3 gamma epitopes SEQ ID NO: 33  93-107 helixLEAVCQDVLSLLDNY SEQ ID NO: 34 191-199 helix EQACHLAKT SEQ ID NO: 35144-155 helix ATVVESSEKAYS SEQ ID NO: 36 144-152 helix ATVVESSEKSEQ ID NO: 37 147-155 helix VESSEKAYS SEQ ID NO: 38 168-177 helixTHPIRLGLAL SEQ ID NO: 39  82-92 helix VRAYREKIEKE SEQ ID NO: 40  68-79helix QKTSADGNEKKI SEQ ID NO: 41 138-146 helix ATGEKRATV SEQ ID NO: 42163-170 helix EHMQPTHP SEQ ID NO: 43 141-146 helix EKRATV SEQ ID NO: 44 69-77 loop KTSADGNEK SEQ ID NO: 45  32-40 loop ELNEPLSNE SEQ ID NO: 46103-117 loop LLDNYLIKNCSETQY SEQ ID NO: 47 130-143 loop YYRYLAEVATGEKRSEQ ID NO: 48 184-194 loop YEIQNAPEQAC SEQ ID NO: 49 206-218 loopAELDTLNEDSYKD SEQ ID NO: 50  44-57 non-helix LLSVAYKNVVGARRSEQ ID NO: 51  75-83 non-helix NEKKIEMVR SEQ ID NO: 52  15-23 non-helixEQAERYDDM SEQ ID NO: 53 130-138 non-helix YYRYLAEVA SEQ ID NO: 54118-125 non-helix ESKVFYLK SEQ ID NO: 55 210-218 non-helix TLNEDSYKDSEQ ID NO: 56  77-84 non-helix KKIEMVRA SEQ ID NO: 57  76-86 non-helixEKKIEMVRAYR SEQ ID NO: 58 142-158 non-helix KRATVVESSEKAYSE AHSEQ ID NO: 59 105-120 non-helix DNYLIKNCSETQYESK SEQ ID NO: 60 237-246non-helix QQDDDGGEGN SEQ ID NO: 61  75-82 non-helix NEKKIEMVSEQ ID NO: 62 104-116 non-helix LDNYLIKNCSETQ

TABLE 3 Protein Sequences of recombinant human14-3-3 eta (SEQ ID NO: 63) and recombinanthuman 14-3-3 gamma (SEQ ID NO: 64) SEQ ID NO: 63MGDREQLLQR ARLAEQAERY DDMASAMKAV TELNEPLSNE  40DRNLLSVAYK NVVGARRSSW RVISSIEQKT MADGNEKKLE  80KVKAYREKIE KELETVCNDV LSLLDKFLIK NCNDFQYESK 120VFYLKMKGDY YRYLAEVASG EKKNSVVEAS EAAYKEAFEI 160SKEQMQPTHP IRLGLALNFS VFYYEIQNAP EQACLLAKQA 200FDDAIAELDT LNEDSYKDST LIMQLLRDNL TLWTSDQQDE 240 EAGEGN SEQ ID NO: 64MVDREQLVQK ARLAEQAERY DDMAAAMKNV TELNEPLSNE  40ERNLLSVAYK NVVGARRSSW RVISSIEQKT SADGNEKKIE  80MVRAYREKIE KELEAVCQDV LSLLDNYLIK NCSETQYESK 120VFYLKMKGDY YRYLAEVATG EKRATVVESS EKAYSEAHEI 160SKEHMQPTHP IRLGLALNYS VFYYEIQNAP EQACHLAKTA 200FDDAIAELDT LNEDSYKDST LIMQLLRDNL TLWTSDQQDD 240 DGGEGNN

In sequences comprising a cysteine residue, in one embodiment, thecysteine residue is replaced by a serine residue to avoid the formationof disulfide bonds. The cysteine may be an internal cysteine residue ora terminal cysteine residue.

Peptide epitopes may be modified for various purposes, includingconjugation to an additional moiety, e.g., conjugation to a moiety toproduce an immunogen comprising the epitope. As will be appreciated,cysteine may be placed appropriately for conjugation to carrier and toprovide for exposure of the area that is desired to be exposed forpurposes of making antibody. In case of KKLE the cysteine was added onto the C-terminal end in order to expose the other side. The carrierused may be quite large and may mask the first few amino acids.

Example 1: 14-3-3 Eta Immunogen Sequences and Anti-14-3-3 Eta Antibodies

To prepare monospecific anti-14-3-3 eta antibodies, various peptides, 8to 15 amino acids in length, were selected based on our own criteria.These peptides, as well as full-length recombinant native (untagged)14-3-3 eta were used as immunogens in the production of monoclonalantibodies. A protein sequence alignment for the 7 isoforms of 14-3-3 isshown in FIG. 4 (14-3-3 gamma (SEQ ID NO: 64, 14-3-3 eta (SEQ ID NO:63),14-3-3 alpha/beta (SEQ ID NO: 71), 14-3-3 zeta (SEQ ID NO:72), 14-3-3theta (SEQ ID NO:73), 14-3-3 sigma (SEQ ID NO:74), and 14-3-3 epsilon(SEQ ID NO:75)).

Immunogen #1: C-LDKFLIKNSNDF (SEQ ID NO:76) (Amino Acid Sequence104-115; “AUG1-CLDK”). A peptide corresponding to a segment of human14-3-3 eta residues 104-115 was modified by addition of an N-terminalcysteine moiety for conjugation to carrier, and replacement of internalcysteine-112 moiety to avoid formation of internal disulphide bonds.

Immunogen #2: KKLEKVKAYR-C (SEQ ID NO:77) (Amino Acid Sequence 77-86;“AUG2-KKLE”). A peptide corresponding to a segment of human 14-3-3 etaresidues 77-86 was modified by addition of a C-terminal cysteine moietyfor conjugation to carrier.

Immunogen #3: C-KNSVVEASEAAYKEA (SEQ ID NO:78) (Amino Acid Sequence143-157; “AUG3-CKNS”). A peptide corresponding to a segment of human14-3-3 eta residues 143-157 was modified by addition of an N-terminalcysteine moiety for conjugation to carrier.

Immunogen #4: Full length human recombinant 14-3-3 eta (SEQ ID NO: 63),Protein Accession #: NP_003396.

Immunization

Groups of 4 female BALB/c mice were initially immunized byintraperitoneal injections using 50 ug of antigen (Immunogen #1, #2, #3or #4) per mouse in Complete Freund's Adjuvant. Four subsequent boostswere administered as above, spaced at 3 week intervals, with antigen inIncomplete Freund's Adjuvant. When the serum titre had risen more than10-fold from that of the pre-immune serum sample, as determined byELISA, the 2 highest responders in each group were each boostedintravenously with 10 ug of antigen in 100 ul of sterile PBS pH 7.4. Thetitrations of serum samples from the immunized mice taken after thesecond boost are shown in FIG. 1 (Immunogen #1; CLDK), FIG. 2 (Immunogen#2; KKLE), FIG. 3 (Immunogen #3; CKNS) and FIG. 8 (Immunogen #4).

Fusion Method

Three days after the final boost, the donor mice were sacrificed and thespleen cells were harvested and pooled. Fusion of the splenocytes withSP2/0 BALB/c parental myeloma cells was performed as previouslydescribed (Kohler et al., infra), except that one-step selection andcloning of the hybridomas was performed. Clones were picked 11 days postfusion and resuspended in wells of 96-well tissue culture plates in: 200μl of D-MEM medium containing 1% hypoxanthine/thymidine, 20% fetalbovine serum, 2 mM GlutaMax I, 1 mM Sodium Pyruvate, 50 μg/mlGentamycin, 1% OPI and 0.6 ng/ml IL-6. After 4 days, the supernatantswere screened by ELISA for antibody activity on plates coated with 1ug/well of purified antigen.

Procedure for Revival of Slow Growing Hybridoma Clones

Hybridoma cell lines that were growing slowly or looked unhealthy couldusually be rescued by the addition of a rich growth media containing:D-MEM medium with 1% hypoxanthine/thymidine, 20% fetal bovine serum, 2mM GlutaMax I, 1 mM Sodium Pyruvate, 50 μg/ml Gentamycin, 1% OPI, 20%conditioned EL-4 tissue culture supernatant and 0.6 ng/ml IL-6. EL-4 isa murine thymoma cell line, which when stimulated with phorbal12-myristate 12-acetate (PMA, from Sigma, cat #P-8139) causes the cellsto secrete interleukin 2 (IL-2), a B cell differentiating factor(EL-BCDF-nak), and two B cell growth factors (BSF-p1 and EL-BCGF-swa)and other additional lymphokines, which greatly enhance lymphocytegrowth and differentiation. See G. Kohler, and C. Milstein, Preparationof monoclonal antibodies, Nature 25 (1975) 256-259; Ma, M., S. Wu, M.Howard and A. Borkovec. 1984. Enhanced production of mouse hybridomas topicomoles of antigen using EL-4 conditioned media with an in vitroimmunization protocol. In Vitro 20:739.

After 30 days of stability testing, a total of 100 viable clones wereobtained that secreted IgG capable of recognizing recombinant 14-3-3eta. For the purposes of identifying lead clones to pursue, the 100viable clones were screened using a series of methods including:immunoblotting (dot blot), a trapping assay and a custom capture(sandwich) ELISA. All 100 clones were also tested for cross-reactivityusing the custom capture (sandwich) ELISA with the other six 14-3-3isoforms.

Example 2 Testing the Cross-Reactivity of Tissue Culture (TC)Supernatants from Hybridoma Clomes Using Biotinylated 14-3-3 Isoforms asBait in a Capture ELISA

We have utilized a custom capture ELISA using the seven 14-3-3 isoformsas “bait” to determine whether any of the hybridoma clones that we haveproduced cross-react or recognize any of the six isoforms other than14-3-3 Eta (η). As is evidenced by the representative data presented inTable 4, four of the selected hybridoma clones (AUG3-CKNS-2D5,AUG3-CKNS-7F8, AUG3-CKNS-7H8, AUG4-ETA-8F10) bind to and recognize14-3-3 Eta at two serial dilutions, but do not bind with or cross-reactwith any of the other 14-3-3 isoforms, even at the lower dilutiontested. This data clearly demonstrates that these clones are highlyspecific for 14-3-3 Eta (η). By contrast, one clone, AUG3-CKNS-4F10,binds with or cross-reacts with three other 14-3-3 isoforms, mainly14-3-3 gamma, beta and zeta respectively. Taken together, these dataindicate that our custom capture ELISA represents an effective methodfor screening and identifying hybridoma clones which are highly specificfor the 14-3-3 Eta (η) isoform.

The Custom Capture ELISA experiment in Table 4 was carried out asfollows. ELISA plates were coated with neat overgrown TC supernatant at100 μL/well and incubated overnight at 4° C. Biotin-labelled 14-3-3(corresponding to all seven isoforms) was titrated from 1/500 to 1/16000overtop and incubated for 1 hour at room temperature. Plates were thenblocked with 3% skim milk powder in PBS (pH 7.4) at 100 μL/well andincubated for 1 hour at room temperature. 1/8000 Streptavidin-HRPO wasdiluted in PBS-Tween, added at 100 μL/well and incubated for 1 hour at37° C. with shaking. TMB buffer was added at 50 μL per well andincubated in the dark at room temperature. Reactions were stopped with50 μL 1M HCl per well after 10 minutes and read at OD450 nm.

TABLE 4 a Testing Cross-reactivity by ELISA Testing the cross-reactivityof tissue culture (TC) supernatants from hybridoma clones usingbiotinylated 14-3-3 isoforms as bait in a capture ELISA (measured atOD₄₅₀ nm) 14-3-3 Isoform: Gamma Beta Sigma Theta/Tau Zeta Epsilon Eta(γ) (β) (σ) (θ) (ξ) (ϵ) (η) TC dilution: super- 1:15 1:30 1:15 1:30 1:151:30 1:15 1:30 1:15 1:30 1:15 1:30 1:15 1:30 natant: 00 00 00 00 00 0000 00 00 00 00 00 00 00 AUG3- 0.076 0.073 0.085 0.075 0.084 0.076 0.1010.082 0.097 0.076 0.074 0.064 0.351 0.263 CKNS- 2D5 *AUG3- 0.084 0.0760.096 0.085 0.125 0.102 0.185 0.153 0.167 0.122 0.139 0.101 0.114 0.09CKNS- 4F10 AUG3- 0.072 0.067 0.078 0.076 0.083 0.076 0.116 0.104 0.0930.084 0.089 0.076 0.946 0.741 CKNS- 7F8 AUG3- 0.07 0.066 0.072 0.0630.087 0.078 0.098 0.083 0.089 0.08 0.074 0.064 0.774 0.608 CKNS- 7H8AUG4- 0.072 0.069 0.073 0.069 0.092 0.084 0.109 0.097 0.099 0.082 0.0990.09 0.169 0.131 ETA- 8F10 pre- 0.097 0.074 0.093 0.081 0.136 0.1130.193 0.158 0.152 0.119 0.144 0.115 0.152 0.11 immune serum (1:250)*control antibody

TABLE 4b Testing Cross-reactivity by ELISA (background (pre-immuneserum) values subtracted out) Testing the cross-reactivity of tissueculture (TC) supernatants from hybridoma clones using biotinylated14-3-3 isoforms as bait in a capture ELISA (measured at OD₄₅₀ nm) 14-3-3Isoform: Gamma Beta Sigma Theta/Tau Zeta Epsilon Eta (γ) (β) (σ) (θ) (ξ)(ϵ) (η) TC dilution: super- 1:15 1:30 1:15 1:30 1:15 1:30 1:15 1:30 1:151:30 1:15 1:30 1:15 1:30 natant: 00 00 00 00 00 00 00 00 00 00 00 00 0000 AUG3- −0.021 −0.001 −0.008 −0.006 −0.052 −0.037 −0.092 −0.076 −0.055−0.043 −0.070 −0.051 0.199 0.153 CKNS- 2D5 *AUG3- −0.013 0.002 0.0030.004 −0.011 −0.011 −0.008 −0.005 0.015 0.003 −0.005 −0.014 −0.038−0.020 CKNS- 4F10 AUG3- −0.025 −0.007 −0.015 −0.005 −0.053 −0.037 −0.077−0.054 −0.059 −0.535 −0.055 −0.039 0.794 0.631 CKNS- 7F8 AUG3- −0.027−0.008 −0.021 −0.018 −0.049 −0.035 −0.095 −0.075 −0.063 −0.039 −0.070−0.051 0.622 0.498 CKNS- 7H8 AUG4- −0.025 −0.005 −5.020 −0.012 −0.044−0.029 −0.084 −0.061 −0.053 −0.037 −0.045 −0.025 0.017 0.021 ETA- 8F10*control antibody

Example 3: 14-3-3 Expression in Synovial Fluid and Serum of RA AffectedPatients

The levels of the different isoforms of 14-3-3 proteins—β, γ, ε, η, τ σand ζ—in pooled patient synovial fluid (SF) and serum (PS) samples wereanalyzed by western analysis using keratinocyte cell lysate (K) as apositive control. Only the η and γ isoforms were detected in SF samples,and stained with greater intensity compared to PS. Articular jointsynovial fluid samples from 17 RA patients who presented with activesynovitis, but had not yet received anti-TNF therapies also exhibitedconsistent expression of the η isoform of 14-3-3 (data not shown). Allpatients had a disease activity score (DAS) greater than 6.0.

Example 4: MMP Expression in Patient Synovial Fluid Serum

To determine if these variations were correlated to those of MMP-1 andMMP-3 in the same synovial samples, a total of 12 RA synovial fluidsamples and their matched serum samples were simultaneously evaluatedfor 14-3-3η and γ as well as for MMP-1 and MMP-3 proteins. 14-3-3η wasdetected in all samples. MMP-1 was detected in all samples, both SF andPS, while MMP-3 was more variable in the levels detected. The 14-3-3γisoform was also detected in patient synovial fluid and serum samples(data not shown).

The expression of MMP-1 and MMP-3 demonstrate significant correlationwith the expression of the 14-3-3η and γ isoforms in both synovial fluidand serum (Table 5).

TABLE 5 Correlation of MMP and 14-3-3 protein levels in serum andsynovial fluid. 14-3-3 η 14-3-3 η 14-3-3 γ 14-3-3 γ serum Synovium serumsynovium MMP-1 r = 0.62; r = 0.83; r = 0.77; r = 0.65; p = 0.02  p =0.03  p = 0.02  p = 0.03  MMP-3 r = 0.68; r = 0.77; r = 0.80; r = 0.76;p = 0.01   p = 0.003  p = 0.03  p = 0.04 

Example 5: Sensitivity of Western Blot Detection of 14-3-3 Protein inPatient Serum and Synovial Fluid Samples

To determine the detection level of 14-3-3η in synovial fluid and serumsamples, samples from 12 RA-affected or normal patients were pooled, andlimiting dilutions of the pooled samples were analyzed by western blot.14-3-3η was detectable over a range of dilutions—as low as 0.1 μleffective volume of synovial fluid and 1.0 μl effective volume of serum(data not shown).

2 μl of pooled normal serum (NS) or patient serum (PS) was run alongsideknown concentrations of recombinant 14-3-3η, ranging from 0.05-2.0 μg.The 2 μl volume of NS and PS samples was estimated to have approximately1-1.5 and 15-20 μg of 14-3-3η, respectively (data not shown). Thissuggests that the level of 14-3-3η occurs in about a 10-fold excess inthe serum of RA affected patients, compared to normal patients.

For more details, and results, see Kilani et al., J. Rheumatology,34:1650-1657, 2007.

Example 6: R-18 Peptide Interacts with Extracellular 14-3-3 Protein andInhibits Induction of MMP-1 Expression Induced by Extracellular 14-3-3Protein

The sequence of biotinylated R18 is as follows:Biotin-Pro-His-Cys-Val-Pro-Arg-Asp-Leu-Ser-Trp-Leu-Asp-Leu-Glu-Ala-Asn-Met-Cys-Leu-Pro-OH(SEQ ID NO:79).

In order to demonstrate the ability of R-18 to block or suppress theMMP-inducing effect of 14-3-3 proteins, we treated subconfluent culturesof dermal fibroblasts with keratinocyte-like cell conditioned medium(KLCCM) that has been demonstrated by mass spectrometry to containseveral 14-3-3 isoforms (unpublished data). The KLCCM used in theseexperiments was taken from day 28 of cell transdifferentiation due totheir high capacity of inducing MMP-1 expression in dermal fibroblasts(data not shown). Some samples of the conditioned media (KLCCM) wereexposed to biotinylated R-18 and avidin sepharose to remove or “pulldown” the 14-3-3 proteins present within the KLCCM.

The results demonstrated that dermal fibroblasts expressed MMP-1, aftertreatment with the KLCCM and that the expression of MMP-1 could bepartially inhibited (68.8% reduction) by pre-treatment of the KLCCM withbiotinylated R-18 and avidin sepharose, which would selectively depletethe KLCCM of 14-3-3 proteins (FIG. 5, Panel A “Pull-down).

Recombinant 14-3-3 sigma (stratifin), which is known to induce MMP-1,was used as positive control at 5 μg/ml (“Pulldown”−) or after exposure(“Pull-down”+). As negative control, dermal fibroblasts treated withmedium that was not conditioned (DMEM (49%), KSFM (49%) plus 2% FBS)were used (“Pull-down”−). Panel B of FIG. 5 shows the densitometricanalysis of MMP-1β-actin ratio. Findings from three independentexperiments exhibited statistical significance (P value: 0.02).

Example 7 Immunoprecipation of Human Recombinant 14-3-3 Eta andEndogenous 14-3-3 Eta from HeLa Cells

Monoclonal anti-14-3-3 antibodies from Example 1 were tested for theirability to immunoprecipitate or “capture” both recombinant andendogenous cellular 14-3-3 eta. For the therapeutic methods of theinvention described herein, it is preferable to use antibodies that havethe ability to immunoprecipitate or recognize 14-3-3 eta in its native3-D configuration. Culture supernatants from anti 14-3-3 eta hybridomaclones were incubated at 4° C. for 2 hours with either buffer containing100 ng human recombinant 14-3-3 eta, or buffer containing supernatant(200 μg protein) from lysed HeLa cells. lmmunoprecipitates werecollected with Protein A/G agarose using standard methodology.lmmunoprecipitates were analysed by SDS-PAGE and Western Blotting. FIG.6 shows a Western Blot obtained using Hybridoma clone 7B11, which wasmade using Immunogen #4 (full length recombinant 14-3-3 eta. Lane 1:Protein NG agarose beads alone; Lane 2: Protein A/G agarose beads weremixed with cell lysate; Lane 3: Protein A/G agarose beads were mixedwith recombinant human 14-3-3 eta; Lane 4: Protein A/G agarose beadswere mixed with hybridoma supernatant; Lane 5: Protein A/G agarose beadswere mixed with hybridoma supernatant and cell lysate; Lane 6: ProteinA/G agarose beads were mixed with hybridoma supernatant and recombinant14-3-3 eta. The data show that clone 7B11 immunoprecipitated both HeLacell-derived 14-3-3 eta (Lane 5) and human recombinant 14-3-3 eta (Lane6).

FIG. 7 shows a Western Blot obtained by using hybridoma clone 2D5 madeagainst Immunogen #3 (CKNS). Lane 1: Protein A/G agarose beads alone;Lane 2: Protein A/G agarose beads were mixed with cell lysate; Lane 3:Protein A/G agarose beads were mixed with recombinant human 14-3-3 eta;Lane 4: Protein A/G agarose beads were mixed with hybridoma supernatant;Lane 5: Protein A/G agarose beads were mixed with hybridoma supernatantand cell lysate; Lane 6: Protein A/G agarose beads were mixed withhybridoma supernatant and recombinant 14-3-3 eta. The data show thatclone 2D5 immunoprecipitated both HeLa cell lysate-derived 14-3-3 eta(Lane 5) and human recombinant 14-3-3 eta (Lane 6).

Similar analyses were performed for several other hybridoma clones (datanot shown). These experiments demonstrate that the monoclonal antibodiesproduced in Example 1 are capable of binding to and immunoprecipitatingor “capturing” 14-3-3 eta in its native configuration, as evidenced bythe immunoprecipitation of the protein from HeLa cell lysates.

Example 8: Anti-14-4-3 Antibody Reduces MMP Expression in Mouse RAModel; 14-3-3 Antagonist Peptide Reduces MMP Expression in Mouse RAModel

Collagen-induced arthritis is induced in Male DBA mice by injection of100 μg of purified type II collagen emulsified in Freund's completeadjuvant at the base of the tail as described in Williams et al., PNAS,89:9784-9788, 1992. Mice are inspected daily thereafter and mice thatexhibit erythema and/or swelling in one of more limbs are assignedrandomly to a treatment regimen with one or more antagonists 14-3-3 etadescribed herein or to a placebo treatment. Alternatively, a treatmentregimen is begun on the day prior to immunization with type II collagen.Various treatment regimens are implemented, using groups of 10 mice, asfollows:

(1) R-18 peptide is administered at various dosages ranging from 0.1 and20 mg/kg (a) intraperitoneally or (b) into the synovium, twice weekly.

(2) Selected anti-14-3-3 eta antibodies obtained and purified from thehybridoma supernatants of Example 1 are administered at various dosagesranging from 0.10 to 20 mg/kg (a) intraperitoneally or (b) into thesynovium, twice weekly.

(3) Placebo treatment

The arthritis is monitored over a 20-day treatment period, and thefollowing disease indices are evaluated.

Clinical score. Mouse limbs are assessed for swelling, erythema, jointrigidity, and paw swelling. The clinical indicia of arthritis is reducedin animals in which the treatment regimen has been efficacious, ascompared to placebo controls.

14-3-3, MMP-1 and/or MMP-3 expression in the synovium. Synovial samplesare taken at various time points, and the 14-3-3, preferably 14-3-3gamma and/or 14-3-3 eta, and MMP-1 and/or MMP-3 levels are determined.The levels of MMP-1 and MMP-3 are reduced in animals in which thetreatment regimen has been efficacious, as compared to placebo controls.

Histopathological assessment. Arthritic paws are fixed, embedded inparaffin, sectioned and stained with hematoxylin and eosin formicroscopic evaluation. The severity of arthritis in each joint isgraded according to the following criteria: mild=minimal synovitis,cartilage loss, and bone erosions limited to discrete foci;moderate=synovitis and erosions present but normal joint architectureintact; severe=synovitis, extensive erosions, and joint architecturedisrupted. The severity of arthritis detected by histopathology isreduced in animals in which the treatment regimen has been efficacious,as compared to placebo controls.

Example 9 Anti-14-4-3 Antibody Reduces MMP Expression in Rabbit RAModel; 14-3-3 Antagonist Peptide Reduces MMP Expression in Rabbit RAModel Induced by Implantation of Cells Secreting IL-1

The 14-3-3 eta antagonists of the invention are evaluated in a rabbitmodel in which arthritis is induced by the implantation of 5×10⁵ IL-1producing cells into the knee joints of New Zealand white rabbits asdescribed in Yao et al., Arthritis Research and Therapy 2006, 8:R16,available on line at http://arthritis-research.com/content/8/1/R16.Testing and evaluation is done essentially as described in Example 8.

Example 10 Anti-14-4-3 Antibody Reduces MMP expression in RA Model;14-3-3 Antagonist Peptide Reduces MMP Expression in RA

Experimental arthritis is induced in Brown Norway rats or in New Zealandwhite rabbits by the injection of recombinant 14-3-3 eta protein intothe synovium of leg joints. Testing and evaluation is done essentiallyas described in Example 8.

Other models of rheumatoid arthritis (collagen-induced arthritis, “CIA”)and experimental designs useful for the methods of the invention can befound for example, in the following references: Williams, Methods MolMed. 2004; 98:207-16. Collagen-induced arthritis as a model forrheumatoid arthritis; Brand, Com. Med., 55:114-122, 2005; Vierboom etal., Drug Discovery Today, 12:327-335, 2007; Sakaguchi et al., Curr.Opin. Immunol., 17:589-594, 2005.

Prior to commencing an initial therapeutic regimen in a particularanimal model, it is preferable to first validate the model as aninflammatory disorder model involving 14-3-3. Preferably, the levels of14-3-3 and MMP, preferably 14-3-3 eta and/or 14-3-3 gamma, andpreferably MMP-1 and/or MMP-3, are determined to show elevationfollowing the induction of experimental arthritis in the model.

General Methods Western Blotting

Samples (synovial fluid or serum (2 μl of each), recombinant human14-3-3 eta, cell lysates or cell-lysate immunoprecipitates) weresubjected to SDS-PAGE analysis with 12-15% (wt/vol) acrylamide gel, andelectrotransferred onto PVDF membranes. Non-specific proteins onmembranes were blocked in 5% skim milk powder in PBS-0.1% Tween-20overnight. Immunoblotting for Example 3 was performed using 2 μg/ml of 7isoforms specific rabbit anti-human 14-3-3 polyclonal antibodies (MartinH, Patel Y, Jones D, Howell S, Robinson K and Aitken A 1993. Antibodiesagainst the major brain isoforms of 14-3-3 protein. An antibody specificfor the N-acetylated amino-terminus of a protein. FEBS Letters.331:296-303). In some experiments, mainly Example 7, the antibodies fromthe hybridoma clones in Example 1 were used for the immunoprecipitationor ‘capture’ experiments. The immunprecipitates were resolved bySDS-PAGE and the membranes were blocked in skim milk and then incubatedwith primary 14-3-3 eta (1:1000, BioMol International SE-486) and thenthe appropriate secondary horseradish peroxidise conjugated anti-rabbitIgG or anti-mouse IgG antibodies (1:2500 dilution). Immunoreactiveproteins were then visualized using the ECL plus western blottingdetection system. Keratinocyte cell lysate (K), recombinant proteinand/or HeLa cell lysate was used as a positive control. SF: synovialfluid; PS: patient serum.

Patient Samples

Synovial fluid was obtained from the knee joints of patients with activesynovitis prior to the institution of anti-TNF therapeutics. Allpatients had a DAS score >6.0. Matched blood samples were obtained bystandard venipuncture procedures. The clot was removed bycentrifugation.

Recombinant 14-3-3 Eta

cDNA for keratinocyte-derived 14-3-3 eta was prepared from total RNAextracted from human keratinocytes, cloned and expressed in E. coli, andaffinity purified, following the methods described in Ghahary et al 2004J Invest Dermatol 122:1188-1197 (REF 36, infra). Primers used for PCRamplification of the 14-3-3 eta cDNA were

(SEQ ID NO: 80) (GCGAATTCCTGCAGCGGGCGCGGCTGGCCGA) and (SEQ ID NO: 81)(GCTCGAGCCTGAAGGATCTTCAGTTGCCTTC).

Untagged Recombinant 14-3-3 Proteins

cDNA was derived from a human source, cloned and expressed in E. coli,and affinity purified. Primers used for the PCR amplification of the14-3-3 eta cDNA were:

(SEQ ID NO: 82) (agaattcagttgccttctcctgctt) and (SEQ ID NO: 83)(acatatgggggaccggga); for 14-3-3 gamma (SEQ ID NO: 84)(agaattcttaattgttgccttcgccg) and (SEQ ID NO: 85) (acatatggtggaccgcgagc);for 14-3-3 beta (SEQ ID NO: 86) (acatatgacaatggataaaagtgagctg) and(SEQ ID NO:87) (agaattcttagttctctccctccccagc); for 14-3-3 epsilon(SEQ ID NO: 88) (acatatggatgatcgagaggatctg) and (SEQ ID NO: 89)(agaattctcactgattttcgtcttccac); for 14-3-3 sigma (SEQ ID NO: 90)(acatatggagagagccagtctgatcc) and (SEQ ID NO: 91)(agaattcagctctggggctcctg); for 14-3-3 theta (SEQ ID NO: 92)(acatatggagaagactgagctgatcc) and (SEQ ID NO: 93)(agaattcttagttttcagccccttctgc); for 14-3-3 zeta (SEQ ID NO: 94)(acatatggataaaaatgagctggttc) and (SEQ ID NO: 95)(agaattcttaattttcccctccttctcct).

ELISA Assay Conditions

For screening and testing: For screening and testing, 1.0 μg/well ofanti-AUG1-CLDK, anti-AUG2-KKLE, anti-AUG3-CKNS or anti-14-3-3 ETAantigen was coated onto ELISA plates in dH₂O at 50 μL/well and drieddown overnight at 37° C. Testing on 14-3-3 ETA antigen 0.25 ug/well wascoated in carbonate coating buffer and incubated at 4° C. overnight.

For testing by antibody trapping assay: 1/10000 Goat anti-mouse IgG/IgMtrapping antibody (Pierce cat #31182) was coated onto ELISA plate incarbonate coating buffer (pH 9.6) at 100 μL/well incubated overnight at4° C.

For testing on negative control antigen: 0.5 μg/well HT (humantransferrin) antigen was coated onto ELISA plate in dH₂O at 50 μL/welland dried down overnight at 37° C.

For testing by Capture ELISA: ELISA plate was coated with neat overgrownTC sup at 100 μL/well incubated overnight at 4° C. Biotin labelled14-3-3 ETA (or one of the six other 14-3-3 family members) was titratedfrom 1/500 to 1/16000 overtop and incubated for 1 hour at roomtemperature.

Blocking: Plates were blocked with 3% skim milk powder in PBS (pH 7.4)at 100 μL/well and incubated for 1 hour at room temperature.

1° antibody. Mouse anti-AUG1-CLDK, anti-AUG2-KKLE, anti-AUG3-CKNS oranti-14-3-3 eta hybridoma tissue culture supernatant and mousemonoclonal controls were added at 100 μL neat per well for screening andtesting. Mouse anti-AUG1-CLDK, anti-AUG2-KKLE, anti-AUG3-CKNS oranti-14-3-3 eta immune serum and mouse pre-immune serum were diluted1/500 in SP2/0 tissue culture supernatant added at 100 μL/well forscreening and testing. Incubated for 1 hour at 37° C. with shaking forboth the screening and testing.

2° antibody used for screening and testing: 1/25000 Goat anti-mouse IgGFc HRP conjugated (Jackson cat #115-035-164) was used in screening andtesting. Secondary antibody diluted in PBS-Tween added at 100 μL/welland incubated for 1 hour at 37° C. with shaking.

Streptavidin used for Capture ELISA: Add 100 ul/well of StreptavidinHRPO (1:8000, CedarLane cat #CLCSA1007) and incubated for 1 hour at roomtemperature with shaking.

Substrate: TMB buffer (BioFx cat #TMBW-1000-01) was added at 50 μL perwell and incubated in the dark at room temperature. Reactions forscreening and testing were stopped with 50 μL 1M HCl per well after 10minutes and read at OD₄₅₀nm.

Dot Blot Conditions

For Screening: Millipore, Immobilon Transfer Membrane cat #IPVH304F0 wasused. 14-3-3 ETA antigen was boiled in sample buffer 5 minutes andallowed to cool. Antigen was dotted on for a total of 6 ug dot amountswith a pipettor. After allowing antigen to dry for 15 minutes blots werewashed with several changes of PBS-Tween pH 7.4. Blots were kept inseparate petri dishes for entire screening process.

Blocking: The PVDF membrane was blocked with 5% milk powder in PBS (pH7.4) for 1 hour at room temperature. Blot was washed after blocking for15 minutes with several changes of PBS-Tween pH 7.4. Blots were allowedto dry on paper towels face up for 10 minutes prior to primary antibodyapplication.

1° antibody: Mouse AUG1-CLDK, anti-AUG2-KKLE, anti-AUG3-CKNS oranti-14-3-3 eta hybridoma tissue culture supernatant and mousemonoclonal controls were incubated with blots in separate petri dishes.Mouse anti-AUG1-CLDK, anti-AUG2-KKLE, anti-AUG3-CKNS or anti-14-3-3 etaimmune and mouse pre-immune sera were diluted 1/500 in SP2/0 tissueculture supernatant used as controls. Blots were incubated with shakingfor 1 hour at room temp. Blots were washed after primary antibodyincubation for 30 minutes with 5 changes of PBS-Tween pH 7.4.

2° antibody: 1/5000 Goat anti-mouse IgG/IgM, (H+L), Alkaline PhosphataseConjugated (Rockland 610-4502) diluted in PBS-Tween pH 7.4 was added tothe blots and incubated with shaking in Petri dishes for one hour atroom temperature. Blots were washed after secondary antibody incubationfor 30 minutes with 5 changes of PBS-Tween pH 7.4. Blots wereequilibrated in Tris 0.1M pH 9 buffer for 10 minutes at room temp andthen dripped dried before addition of substrate.

Substrate: BCIP/NBT developer 1 component AP membrane substrate (BioFXproduct #BCID-1000-01) was dripped onto blot neat at room temp. Thereaction was stopped after 5 minutes with cold tap water and resultswere determined quantitatively by eye and given a score of strongpositive +++, moderate positive ++, weak positive +, slight positive+/−, negative −.

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1-80. (canceled)
 81. A method of reducing expression of matrixmetalloprotease 1 (MMP-1) in the synovium of a subject, comprisingadministering to said subject a 14-3-3 eta antibody that specificallybinds to an epitope of 14-3-3 eta having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 3, 4, 5, 24, 29, 30, 31 and 32.82. The method of claim 81, wherein said antibody is a monoclonalantibody.
 83. The method of claim 81, wherein said monoclonal antibodyis a humanized monoclonal antibody.
 84. The method of claim 81, whereinsaid anti-14-3-3 eta antibody discriminates between 14-3-3 proteinisoforms.
 85. The method of claims 81, wherein said anti-14-3-3 etaantibody specifically binds to a peptide consisting of the amino acidsequence NSVVEASEAAYK (SEQ ID NO: 3) or NSVVEASEA (SEQ ID NO: 4). 86.The method of claim 81, wherein said anti-14-3-3 eta antibodyspecifically binds to a peptide consisting of the amino acid sequenceLDKFLIKNSNDF (SEQ ID NO:30).
 87. The method of claim 81, wherein saidanti-14-3-3 eta antibody is specifically binds to a peptide consistingof the amino acid sequence KKLEKVKAYR (SEQ ID NO:31).
 88. The method ofclaim 81, wherein said anti-14-3-3 eta antibody specifically binds to apeptide consisting of the amino acid sequence KNSWEASEAAYKEA (SEQ IDNO:32).
 89. The method of claim 81, wherein said anti-14-3-3 etaantibody specifically binds to a peptide consisting of the amino acidsequence KKNSVVEASEAAYKEAF (SEQ ID NO:24).
 90. The method of claim 81,wherein said anti-14-3-3 eta antibody specifically binds to a peptideconsisting of the amino acid sequence VEASEAAYK (SEQ ID NO:5).
 91. Themethod of claim 81, wherein said antibody is produced by a hybridomaselected from the group consisting of AUG3-CKNS-2D5, AUG3-CKNS-7F8,AUG3-CKNS-7H8, and AUG4-ETA-8F10.